Chemosensitizing with liposomes containing oligonucleotides

ABSTRACT

The invention relates to the use of oligonucleotide containing cationic liposomal formulations to enhance the efficacy of chemotherapy and/or radiotherapy, particularly as a means to sensitize cancerous tumor tissues to the efficiencies of chemotherapy. This is particularly advantageous in the context of treating raf expressing tumors such as breast, lung, pancreatic and prostate tumors.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/538,241filed on Mar. 30, 2000, now U.S. Pat. No. 6,359,129 which is acontinuation-in-part of U.S. Ser. No. 09/354,109, filed Jul. 15, 1999,now abandoned which is in turn a divisional of U.S. Ser. No. 08/957,327,filed Oct. 24, 1997, now U.S. Pat. No. 6,126,965 which claims benefit ofpriority to Provisional Application Ser. No. 60/041,192, filed Mar. 21,1997. All of these applications are incorporated by reference in theirentirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under grant numbersCA46641, CA68322, CA58984, CA65012, CA52066, and CA74175 awarded by NIH.

FIELD OF THE INVENTION

This invention is related to novel methods of sensitizing tumor tissueto therapy, preferably chemotherapy or a combination of chemotherapy andradiotherapy using a cationic liposomal composition containing anoligonucleotides or combination of oligonucleotides that specificallybinds to a gene expressed by the tumor tissue.

BACKGROUND OF THE INVENTION

The use of chemotherapeutics of treat cancer is well established.Examples of chemotherapeutics finding established application in thetreatment of cancers include by way of examples tamoxifen, toremifene,cisplatin, methotrexate, adriamycin, to name but a few. Often suchchemotherapeutics are utilized in combination, i.e., as coctails inchemotherapeutic regimens, and often in combination with other types oftherapies, e.g., radiation, surgery or antibody-based therapeutics.

While chemotherapeutics have had success in treating a number ofdifferent types of cancers, e.g., some leukemia, breast cancer andprostate cancer, chemotherapy is fraught with problems. For example,chemotherapeutics exhibit toxicity to non-targeted tissue, e.g., theymay cause nephrotoxicity. Another prevalent problem with chemotherapy isthat tumor tissues may become resistant to a particularchemotherapeutic. For example, it is known that some tumors becomeresistant to cisplatin.

In order to alleviate such problems, it is known to administerchemotherapeutics in combination thereby minimizing the risk that thetumor will become resistant to the chemotherapeutic regimen. However,this solution is less than satisfactory, as it does not eliminate thefact that some tumors should become resistant to chemotherapy. This isdisadvantageous as it produces the efficiency of such chemotherapeutic,e.g., requiring that they be administered in greater dosages to elicitcytotoxicity. This is problematic as the risk of systemic toxicity tonon-targeted tissues increases. Also, it may significantly increase thecost of treatment.

It is similarly known that tumors may become resistant to ionizingradiation. For example, it has been reported that tumor resistance maybe correlated with the expression of certain oncogenes such as ras, raf,cot, mos and myc as well as growth factors such as PDGF and FGF, amongothers.

The use of oligonucleotides for treatment of cancer has also beenreported, in particular antisense oligonucleotides that target oncogenesor other genes expressed by the particular cancer. However, antisensetherapy is also subject to some problems that inhibit efficacy,particularly the fact that such oligonucleotides can be unstable in vivoand, therefore, may become degraded before they reach the target site,e.g., tumor cell or viral infected cell.

Attempts to increase the potency of oliogs have included the synthesisof several analogs, with modifications directed primarily to thephosphodiester backbone. For example, phosphorothioate oligonucleotideshave demonstrated enhanced resistance to nuclease digestion. Othermodifications to oligonucleotides have included derivatization withlipophilic moieties such as cholesterol, and polylysine to enhancecellular uptake. Alternatively, the polyanionic nature of the moleculehas been eliminated in methylphosphonate analogs.

Another reported approach has involved the use of cationic liposomes toenhance delivery. Bennet et al., Mol. Pharmacol., 4:1023–1033 (1992).Zelphati et al., J. Lipsome Res., 7(1):31–49 (1997); Thierry et al.,Biochem. Biophys. Res. Comm., 190(3):952–960 (1993). It is widelyaccepted that cationic liposomes must contain enough charge toneutralize the negatively charged oligonucleotides as well as providingenough residual positive charge to the complex to facilitate interactionwith a negatively charged cell surface. (Litzinger et al., J. LiposomeResearch, 7(1): 51–61 (1997)). However, problems associated withprevious cationic liposomal delivery systems similarly includeserum-instability, undesirable biodistribution, andtarget-non-specificity, which hinder their use for efficient nucleicacid delivery in vivo.

Therefore, methods for improving treatment of chemoresistant tumorswould be highly beneficial.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the invention to sensitize tumor tissues to theeffects of chemotherapy by the administration of at least oneoligonucleotide that targets a gene expressed by the tumor tissues as anadjunct to chemotherapy.

It is a more specific object of the invention to sensitize tumor tissuesto the effects of chemotherapy by the administration of at least oneantisense oligonucleotide comprised in a serum stable cationic liposomaldelivery system, wherein the oligo containing cationic liposomes may beadministered prior, during or after chemotherapy.

It is a more specific object of the invention to use as the cationicliposomal delivery system liposomes having enhanced serum stability andtargeting capability that comprise dimethyldioctadecyl ammonium bromide(DDAB), phosphatidylcholine (PC), and cholesterol (CHOL) containing atleast one oligonucleotide that targets a gene expressed by the tumortissue, to chemosensitize tumor tissues to the effects of chemotherapy.

It is another specific object of the invention to use as thechemosensitizing cationic liposomal delivery system cationic liposomesthat comprise the cationic lipid 1,2-dimyristoyl-3-trimethyl ammoniumpropane (DOTAP); phosphatidylcholine (PC), and cholesterol (CHOL),having encapsulated therein at least one oligonucleotide that isdirected adjunct a gene expressed by the tumor tissue.

It is another object of the invention to use as the chemosensitizingcationic delivery system cationic liposomes comprising at least onecationic lipid selected from: 1,2-dioleoyl-3-trimethyl ammonium propane(DATAP), N-(2,3-(dioleoyloxy)propyl)-N,N,N-trimethyl ammonium chloride,or 1-[2-(9(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)heptadecenyl)-3-(2-hydroxyethyl)-imidazolinium chloride)phosphatidylcholine (PC) and cholesterol (CHOL).

It is an even more specific object of the invention to use as thechemosensitizing cationic delivery system cationic liposomes comprising1,2-dimyristoyl-3-trimethyl ammonium propane (DMTAP);phosphatidylcholine (PC), and cholesterol, wherein the respective molarratios range from 0.5 to 1.4; 2.0 to 4.0; and 0.5 to 2.5; and morepreferably 0.75 to 1.25; 3.0 to 4.0; and 1.0 to 2.0; and most preferablyabout 1:3.2:1.6, again containing at least one oligonucleotide.

It is another specific object of the invention to produce achemosensitizing formulation comprised of cationic liposomes comprisingdimethyldioctadecyl ammonium bromide (DDAB), phosphatidylcholine (PC)and cholesterol, wherein the respective molar ratios are 0.5 to 1.5; 2.0to 4.0, and 0.5 to 2.5; more preferably 0.75 to 1.25; 3.0 to 4.0; and1.0 to 2.0; and most preferably about 1:3.2.1.6, again containing atleast one oligonucleotide that targets a gene expressed by the tumor.

It is another specific object of the invention to utilize cationicliposomes comprising at least one cationic lipid selected from:1,2-dimyristoyl-3-trimethyl ammonium propane (DMTAP),dimethyldioctadecyl ammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium propane, (DOTAP) N-[2,3-(dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and1-[2-(9-(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)heptadencenyl)-3-(2-hydroxyethyl)-imidazoliumchlorine; phosphatidylcholine and cholesterol, wherein the molar ratioof total cationic lipid, phosphatidylcholine, and cholesterol preferablyranges from 0.5 to 1.5; 2.0 to 4.0; and 0.5 to 2.5; more preferably 0.75to 1.25; 3.0 to 4.0; and 1.0 to 2.0; and most preferably 0.8 to 1.2; 3.0to 3.5; and 1.4 to 1.8 as a vehicle for in vivo delivery of one or moreoligonucleotides, to sensitize tumors, particularly, chemoresistanttumors to the effects of a particular chemotherapeutic regimen. Thisoligonucleotide may be in the sense or antisense orientation relative toa gene target, expressed by the tumor tissue e.g., an oncogene. Mostpreferably the oligonucleotide will be an antisense oligonucleotide,preferably having a serum ranging from about 8 to 100 nucleotides, morepreferably 15 to 40 nucleotides.

It is an even more specific object of the invention to use the subjectcationic liposomes containing at least one oligonucleotide tochemosensitize solid tumors and cancers including head and neck cancer,prostate cancer, pancreatic cancer, breast cancer, lung cancer, kidneycancer, ovarian cancer, brain cancer, esophageal cancer, sarcoma,carcinoma, myeloma, bladder cancer, liver cancer, colon cancer, penilecancer, B and T cell lymphomas, leukemias, testicular cancer, bonecancer, and other hematologic cancers to effects of chemotherapy.

It is another specific object of the invention to administer antisenseoligonucleotides corresponding to portions of oncogenes preferablyselected from the group consisting of ras, raf, cot, mos, myc,preferably c-raf-1, or a growth factor PDGF, FGF, EGF), as an adjunct tochemotherapy and optionally radiotherapy in order to sensitize cancercells to the effects of the chemotherapy and also potentiallyradiotherapy. Preferably, such oligonucleotides will be administeredusing the subject cationic liposomal cationic liposomal delivery systemsdiscussed supra. The bases comprised in said oligonucleotide may bemodified or unmodified, and the size of such oligonucleotides willpreferably range from 8 to 100 nucleotides; more preferably 12 to 60nucleotides, most preferably from 15 to 40, or 15 to 25 nucleotides.

It is an even more specific object of the invention to administeroligonucleotides comprising 5′-GTG-CTCCATTGATGC-3′ (SEQ ID NO:1) and/or5′-CCTGTATGTGCTCCATT-GATGCAGC-3′ (SEQ ID NO:2), preferably encapsulatedin a cationic liposome, wherein the bases of said oligonucleotides maybe modified or unmodified, as an adjunct to chemotherapy. It is anotherspecific object of the invention to chemosensitize tumor tissue tochemotherapeutic agent(s) by the administration of a cationic liposomecontaining at least one oligonucleotide, preferably an anti-senseoligonucleotides corresponding to a surface or internal antigen oroncogene expressed by the tumor, prior, concurrent or afteradministration of the chemotherapeutic.

In a more specific embodiment the cationic liposomal delivery systemwill comprise a raf anti-sense oligonucleotide, the cancer treated willbe prostate cancer and the chemotherapy will be a regimen that iseffective against prostate cancer.

In another specific embodiment of the invention the oligonucleotideswill be chemically modified, e.g., phosphorothiotated.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1: A liposomal encapsulation of ATG-AS raf ODN (SEQ ID NO:1),5′-fluorescein-labeled ATG-AS raf ODN (SEQ ID NO:1) was encapsulated inliposomes (PC/CHOL/DDAB) as explained in the experimental procedures.Bright field microscopy of blank liposomes (a), and fluorescencemicroscopy of blank liposomes (b) and liposomes containing thefluorescein-labeled ATG-AS raf ODN (SEQ ID NO:1) (C-H) are shown.

FIG. 2: (a) Time-course of cellular uptake of LE-ATG-AS raf ODN (SEQ IDNO:1), SQ-20B cells were incubated with ³²P-5 end labeled and an excessof unlabeled LE-ATG-AS raf ODN (SEQ ID NO:1) (10 μM) for indicated timesas described in the experimental procedures. Results are mean±s.d fromtwo independent experiments each performed in duplicate, (b)Intracellular stability of LE-ATG-AS raf ODN (SEQ ID NO:1). Cells wereincubated with ³²P-end labeled and an excess of unlabeled LE-ATG-AS rafODN (SEQ ID NO:1) (10 μM in 1% FBS containing medium for 1 hour, washedwith PBS and then incubations continued in 20% FBS containing medium for0 hour (lane 2), 2 hours (lane 3), 8 hours (lane 4), 12 hours (lane 5),and 24 hours (lane 6). Cells were lysed and ODNs in various samples wereanalyzed by denaturing gel electrophoresis as explained in theexperimental procedures. Lane 2, radiolabeled control LE-ATG-AS raf ODN(SEQ ID NO: 1); 15-mer, ATG-AS raf ODN (SEQ ID NO:1) migrations ofxylene cyanol (XC) and bromophenol blue (BP) are indicated.

FIG. 3: The plasma concentration-time profile of LE-ATG-AS raf ODN (SEQID NO:1), 30 mg/kg LE-ATG-AS raf ODN (SEQ ID NO:1) (top panel) or TG-ASraf ODN (SEQ ID NO:1) (middle panel) was administered i.e. in Balb/dnu/nu mice. Blood samples were collected from retro-orbital sinus asindicated times after injection and the ODNs in plasma samples wereextracted and analyzed by denaturing gel electrophoresis as explained inthe experimental procedures. St standards prepared by spiking knownamounts of ATG-AS raf ODN (SEQ ID NO:1) in blank plasma. Top panel:Samples were diluted before electrophoresis as follows: lane 1, 12×;lanes 2 and 3, 4×; lanes 4 and 5, 3.3×; lane 6, 2×; lane 7, 1.4×; lane8, 1.3×; and lane 9, I×. St lanes represent 0.25, 0.5 and 1.0 μg/ml ofATG-AS raf ODN (SEQ ID NO:1). 1.0 μg/ml of the standard samplecorresponds to 18.4 μg of ODN. Additional standards (1.4 to >2 logrange) were used to determine ODN concentration over a 24 hour period(data not shown). Middle panel: Samples were diluted before loading asfollows: lane 1, 4×, lanes 2 and 3, 2×, lanes 4, 5 and 6, I×; lane 7,0.75×, lanes 8 and 0, 0.6×. St lanes represent 0.125, 0.25, and 0.5μg/ml of ATG-AS raf ODN (SEQ ID NO:1), 0.5 μg/ml of the standard samplecorresponds to 6.9 ng of ODN. Bottom panel: Plasma concentration-timecurve of LE-ATG-AS raf ODN (SEQ ID NO:1) shown in the top panel.Quantification data were calculated based on comparison with knownconcentrations of the standard samples, and then normalization againstsample dilution factors used for loading.

FIG. 4: Tissue distribution profiles of LE-ATG-AS raf ODN (SEQ ID NO:1).Tissue samples were collected at indicated times after i.v.administration of 30 mg/kg LE-ATG-AS raf ODN (SEQ ID NO:1) ODNs wereextracted from homogenized tissues and probed with ³²P labeled ATG-S rafODN (SEQ ID NO:3) as explained in the experimental procedures, (a)Representative autoradiographs from liver and kidney, (b) ATG-AS raf ODN(SEQ ID NO:1) concentration in different tissues at indicated timesafter a dose of 30 mg/kg LE-ATG-AS raf ODN (SEQ ID NO:1) wasadministered i.v. Quantification data were calculated based oncomparison with known concentrations of the standard samples, and thennormalization against the weights of organs collected.

FIG. 5: Specificity of inhibition of Raf-1 protein expression byLE-ATG-AS raf ODN (SEQ ID NO:1), (a) Time-course analysis,Logarithmically growing SQ-20B cells were treated with 10 μM ofLE-ATG-AS raf ODN (SEQ ID NO:1) (AS), or LE-ATG-S rad ODN (SEQ ID NO:3)(S) for indicated times in 1% FBS containing medium. Untreated controlcells (C) or cells treated with blank liposomes (10 μm) weresimultaneously switched to 1% FBS containing medium for eight hours.Whole cell lysates were normalized for total protein content andimmunoprecipitated with agarose-conjugated polyclonal anti-Raf-1antibody (Santa Cruz). Immune complexes were immunoblotted withpolyclonal anti-RAF-1 antibody as described in the experimentalprocedures (top). Results from three independent experiments werequantified and data are expressed relative to the level of Raf-1 inLE-ATG-S raf ODN-treated (SEQ ID NO:3) cells (bottom), (b) Dose-responseanalysis, Logarithmically growing SQ-20B tumor cells were treated withindicated concentrations of LE-ATG-AS raf ODN (SEQ ID NO:1) (AS) orLE-ATG-S raf ODN (SEQ ID NO:3) (S) in 1% FBS containing medium for eighthours. Normalized cell lysates were analyzed for RAF-1 expression (top).Quantification data from three independent experiments are expressedrelative to the level of Raf-1 in LE-ATG-S raf ODN-treated (SEQ ID NO:3)cells (bottom).

FIG. 6: Inhibition of Raf-1 protein kinase activity by LE-ATG-AS raf ODN(SEQ ID NO:1), Logarithmically growing SQ-20B cells were treated with120 μm LE-ATG-AS raf ODN (SEQ ID NO:1) (AS), or 10 um LE-ATG-S raf ODN(S) (SEQ ID NO:3) for eight hours in 1% FBS containing medium. Controlcells (C) were simultaneously switched to 1% FBS containing medium foreight hours. Whole cell lysates were normalized for protein content, andRaf-1 phosphotransferase activity was assayed in vitro using itsphysiologic substrate, MKK1 as described in the experimental procedures.Radiolabeled reaction products were separated by electrophoresis, andautoradiographed (inset). Quantification data from two independentexperiments, each performed in duplicate, are expressed as Raf-1enzymatic activity in LE-ATG-AS raf ODN (SEQ ID NO:1) treated cellsrelative to LE-ATG-S raf ODN-treated (SEQ ID NO:3) cells.

FIG. 7: Effects of intravenous administration of LE-ATG-AS raf ODN (SEQID NO:1) our Raf-1 expression in normal and tumor tissues. Raf-1expression was examined in liver, kidneys and SQ-20B tumor xenograft ofBalb/c nu/nu mice after i.v. injections of 6 mg/kg daily dose ofLE-ATG-AS raf ODN (SEQ ID NO:1) (AS) or LE-ATG-S raf ODN (SEQ ID NO:3)(S) for five consecutive days. Control mice (C) received normal saline.Representative data showing Raf-1 expression in lysates normalized forprotein content by immunoprecipitation and immunoblotting (top).Quantification data are shown as mean±s.d. from three mice (bottom).

FIG. 8: Inhibition of Raf-1 protein expression in SQ-20B tumorxenografts by intratumoral administration of LE-ATG-AS raf ODN (SEQ IDNO:1): Each animal received intratumoral injections of LE-ATG-AS raf ODN(SEQ ID NO:1) (AS) on the right flank and LE-ATG-S raf ODN (SEQ ID NO:3)(S) on the left flank at a dose of 4 mg/kg daily for seven days asexplained in the experimental procedures. Raf-1 expression in the right(AS) and left (S) tumor xenografts from two representative animals isshown (inset). Quantification data shown are mean±s.d., from threerepresentative mice.

FIG. 9: Antisense sequence-specific inhibition of Raf-1 expression inSQ-20B cells. (A) Logarithmically growing SQ-20B cells were treated withindicated ODN concentrations of LE-5132 (SEQ ID NO:4) (lanes 3, 5 and10), 5132 (SEQ ID NO:4) (lanes 4 and 6), or LE-10353 (SEQ ID NO:5) (lane9) as described in Materials and Methods. Control cells were either leftuntreated (lanes 1 and 7) or treated with 1 uM blank liposomes (BL)(lanes 2 and 8). Whole cell lysates were normalized for total proteincontent and immunoprecipitated with agarose-conjugated polyclonalanti-Raf-1 antibody. Immune complexes were resolved by 7.5% SDS-PAGE andimmunoblotted with polyclonal anti-Raf-1 antibody. (B) Data from threeindependent experiments were quantified and expressed relative to thelevel of Raf-1 in untreated cells.

FIG. 10: Effect of LE-5132 (SEQ ID NO:4) on coagulation time. Normalhuman plasma was mixed with indicated concentration of LE-5132 (SEQ IDNO:4) or 5132 (SEQ ID NO:4) or left untreated and incubated with APTTreagent (purified rabbit brain cephalin extract with allergic acidactivator) for one minute at 37° C. The coagulation reaction wasinitiated by adding calcium chloride, and the time required to formvisible clot be recorded mutually in seconds. Data represents mean=SDfrom three experiments.

FIG. 11: The plasma concentration-time profile of LE-5132 (SEQ ID NO:4)and 5132 (SEQ ID NO:4); 30 mg/kg LE-5132 (SEQ ID NO:4) or 5132 (SEQ IDNO:4) was administered i.v. in Balb/c nu/nu mice. Blood samples werecollected from the retroorbital sinus at indicated times afterinjection, and ODN in plasma samples was extracted and analyzed bydenaturing gel electrophoresis as described. S1, S2 and S3, standardsprepared by spiking known amounts of 5132 (SEQ ID NO:4) in blank plasma.(A) Samples were diluted before electrophoresis as follows: lane 1, 15X;lane 2, 10X; lanes 3 and 4, 5X; lane 5, IX; lanes 6–9, 5 0.8X. (B)Samples were diluted before electrophoresis as follows: lane 1, 15X;lane 2, 10X; lanes 3 and 4, 5X; lane 5, IX; lanes 6–9, 0.8X; S1, S2, andS3 represent 0.25, 0.5, and 1.0 μg/ml of 5132 (SEQ ID NO:4),respectively; 1.0 μg/ml of the standard sample corresponds to 20 ng ofODN. (C) Plasma concentration-time curve of LE-5132 (SEQ ID NO: 4) and5132 (SEQ ID NO:4). Quantification data were calculated based oncomparison with known concentrations of the standard samples and thennormalized against sample dilution factors used for loading.

FIG. 12: Normal tissue pharmacokinetics of LE-5132 (SEQ ID NO:4)/5132(SEQ ID NO:4) as a function of area under the concentration-time curve(AUC). Tissue samples were collected between 0 and 48 hours afteradministration of 30 mg/kg LE-5132 (SEQ ID NO:4) or 5132 (SEQ ID NO:4)as in FIG. 11. ODN were extracted from homogenized tissues and probedwith (³²P)-labeled sense raf ODN. Quantification analysis was performedbased on comparison with known concentration of the standard samples andthen normalized against the weights of the organs collected.

FIG. 13: Effect of LE-5132 (SEQ ID NO:4) on SW-20B tumor growth. SQ-20Brumor cells (2×10⁶) were injected a.c. into the left flank region ofeach male Balb/c nu/nu mouse, 10–12 weeks old. Tumor xenografts weregrown to a mean tumor volume of 94±6.4 mm³, and the animals wererandomized into two treatment groups. Day 0 represents the first day oftreatment. Mice were given i.v. 6 mg/kg LE-5132 (SEQ ID NO:4) or blankliposomes (BL) once daily for the first 7 days, followed by sixadditional doses on alternate days, as indicated by the arrows. Theanimals were killed on day 30. The data shown are mean±SE of 5–7 animalsper group.

FIG. 14: Effect of LE-5132 (SEQ ID NO:4) on Raf-1 protein level inSQ-20B tumors. (A) Tumor-bearing mice were treated with LE-5132 (SEQ IDNO:4) (i.v., 10 mg/kg) or IR (3.8 Gy/day) or both, as explained in thelegend to FIG. 7. Tumors representing various treatment groups wereexcised on day 7 (lanes 1 and 2) and day 14 (lanes 3–8) of treatment.Raf-1 expression was detected in tissue homogenates normalized forprotein content by immunoprecipitation, followed by immunoblotting. Lane1, untreated control; lanes 2 and 3, BL; lane 4, R; lanes 5 and 6,LE-5132 (SEQ ID NO:4)); lanes 7 and 8, LE-5132 (SEQ ID NO:4)+IR, (B)Quantification data shown are mean±SE from 2 animals.

FIG. 15: Effect of LE-5132 (SEQ ID NO:4) and ionizing radiation onSQ-20B rumor growth. (A) SQ-20B tumor xenografts were grown in maleBalb/c nu/nu mice as described. Animals bearing a mean tumor volume of72.0±4.3 mm³ were randomized into five treatment groups. Day 0represents the first day of treatment. Mice were treated with LE-5132(SEQ ID NO:4) 10 mg/kg i.v. (LE-5132 (SEQ ID NO:4)), ionizing radiationonce a day with 3.8 Gy (IR), or a combination of these two treatmentsfor the indicated days (LE-5132 (SEQ ID NO:4)+IR). Control groupsreceived either blank liposomes (BL) or no treatment (C). Animals werekilled on day 45. The data shown are mean±SE of 5–7 animals per group.(B) Fold change in mean tumor volumes in different treatment groups onday 30. The data shown are mean±SE from two independent experiments. 5–7animals per group per experiment. ⁼p<0.001.

FIG. 16: Histopathology of SQ-20B rumors. Tumors were excised 24 hoursafter the final treatment. Shown are examples of the histopathology ofan untreated tumor (A) and tumor treated with LE-5132 (SEQ ID NO:4) (B),IR (C), or LE-5132 (SEQ ID NO:4)+IR (D). x250.

FIG. 17: contains in vitro results of dose-response uptake experimentusing unlabeled antisense raf oligo (ATG-AS (SEQ ID NO:1)) in free(ATG-AJ^(c) (SEQ ID NO:1)) or liposome (DMTAP=PC=CHOL) encapsulated from(LE-ATG-AS (SEQ ID NO:1)).

FIG. 18: contains results of time-course uptake experiment in SQ-20Btumor cells using free (ATG-AS) (SEQ ID NO:1) or liposome encapsulate(LE-ATG-AS (SEQ ID NO:1)) raf oligonucleotides.

FIG. 19: contains half of stability experiment comparing stability ofraf oligonucleotides in free (ATG-AS (SEQ ID NO:1)) or liposomeencapsulate (LE-ATG-AS (SEQ ID NO:1)) μm.

FIG. 20: contains results of another stability experiment comparingstability of free raf oligonucleotides (ATG-AS) (SEQ ID NO:1) orliposome encapsulate (LE-ATG-AS (SEQ ID NO:1)).

FIG. 21: contains survival data in mice/CD2F1 mice administeredDMTAP:PC:CHOL liposomes injected i.v.

FIG. 22 contains survival data from mice CD2F1 mice administered rafoligonucleotides encapsulate in DMTAP:PC:CHOL liposome (LE-ATG-AS (SEQID NO:1)).

FIG. 23: Antitumor efficacy of LEraf Aon (SEQ ID NO:1) in combinationwith cisplatin in athymic nu/nu mice bearing human prostate cancer(PC-3) xenografts. Male athymic mice were inoculated subcutaneously(s.c) with 5×10⁶ PC-3 cells in 0.2 ml phosphate buffered saline/animal.Tumor growth was monitored twice a week until the tumor volumes were 60mm³ to 100 mm³. Animals were randomized into five treatment groupsindicated above (n=8). Mice were treated with indicated dosesintravenously via the tail vein, and the tumor sizes were monitoredtwice a week. Values shown are mean±s.d.

FIG. 24: Antirumor efficacy of LEraf AON (SEQ ID NO:1) in combinationwith epirubicin in athymic nu/nu mice bearing human prostate cancer(PC-3) xenografts. Male athymic mice were inoculated s.c. with 5×10⁶PC-3 cells in 0.2 ml phosphate buffered saline/animal. Tumor growth wasmonitored twice a week until the tumor volumes were 60 mm³ to 100 mm³.Animals were randomized into five treatment groups indicated above(n=6). Mice were treated with indicated doses intravenously via the tailvein, and the tumor sizes were monitored twice a week. Values shown aremean±s.d.

FIG. 25: Antitumor efficacy of LEraf AON (SEQ ID NO:1) in combinationwith mixotantrone in athymic nu/nu mice bearing human prostate cancer(PC-3) xenografts. Male athymic mice were inoculated s.c. with 5×10⁶PC-3 cells in 0.2 ml phosphate buffered saline/animal. Tumor growth wasmonitored twice a week until the tumor volumes were 60 mm³ to 100 mm³.Animals were randomized into five treatment groups indicated above.(n=5) Mice were treated with indicated doses intravenously via the tailvein, and the tumor sizes were monitored twice a week. Values shown aremean±s.d.

FIG. 26A: Antitumor efficacy of a combination of liposome-entrapped rafantisense oligodeoxyribonucleotide (LErafAON (SEQ ID NO:1)) and Gemzar(NDC 0002+7501+01), Gemcitabine HCl, Eli Lilly and Company,Indianapolis, Ind.) in athymic mice bearing human pancreatic carcinomaxenografts (Colo357). Athymic mice were inoculated with approximately1.0×10⁶ Colo357 human pancreatic cancer cells. Tumor volumes wereallowed to reach 50 to 100 mm³ in size. Tumor bearing mice were randomlygrouped into five treatment categories (n=5). LErafAON (SEQ ID NO:1)formulation was prepared as described before (Gokhale et al., GeneTherapy 4, 1289–1299, 1997; and Gokhale et al., manuscript inpreparation). Anticancer drag Gemzar (200 mg vials) was purchased fromoncology pharmacy at Georgetown University Hospital and reconstituted innormal saline (12.5 mg/ml). LErafAON (SEQ ID NO:1) (25.0 mg/kg) orGemzar (750 mg/kg) was injected intravenously on indicated days. Tumorvolumes were measured twice a week, and tumor sizes (% initial) invarious treatment groups were plotted. As shown in this figure, asignificant tumor growth arrest was noted in the combination treatmentgroup (LErafAON (SEQ ID NO:1) plus Gemzar) as compared to single agenttreatment groups (LErafAON (SEQ ID NO:1) and Gemzar) or control groups(N.S. normal saline, BL, Blank liposomes). Values shown are mean±s.d.

FIG. 26B: Antitumor efficacy of a combination of liposome-entrapped rafantisense oligodeoxyribonucleotide (LErafAON (SEQ ID NO:1)) and Gemzar(NDC 002+7501+01, Gemcitabine HCl, Eli 5 Lilly and Company,Indianapolis, In) in athymic mice bearing human pancreatic carcinomaxenografts (Aspc-1). Athymic mice were inoculated with approximately2.5×10⁶ Aspc-1 human pancreatic cancer cells. Tumor volumes were allowedto reach 50 to 100 mm³ in size. Tumor bearing mice were randomly groupedinto five treatment categories (n=5). LErafAON (SEQ ID NO:1) formulationwas prepared as described before (Gokhale et al., Gene Therapy 4,1289–1299, 1997, and Gokhale et al., manuscript in preparation).Anticancer drug Gemzar (200 mg vials) was purchased from oncologypharmacy at Georgetown University Hospital and reconstituted in normalsaline (12.5 mg/ml). LErafAON (SEQ ID NO:1) (25.0 mg/kg) or Gemzar(100.0 mg/kg) was injected intravenously on indicated days. Tumorvolumes were measured twice a week, and tumor sizes (% initial) invarious treatment groups were plotted. As shown in this figure, asignificant tumor growth arrest was noted in the combination treatmentgroup (LErafAON (SEQ ID NO:1) plus Gemzar) as compared to a single agenttreatment groups (LErafAON (SEQ ID NO:1) and Gemzar) or control groups(N.S. normal saline, BL, Blank liposomes). Values shown are mean±.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of a cationic liposomalformulations containing at least one oligonucleotide targeted against agene expressed by a tumor e.g., a solid tumor, to sensitize the tumoragainst the effects of a chemotherapeutic regimen. In earlierapplications, incorporated by reference infra, the inventorsdemonstrated that a novel cationic liposomal delivery system renderedtumor cells more sensitive to the effects of radiotherapy. Thisapplication is on extension of that discovery, as it has now been shownthat this same cationic liposomal delivery system renders tumor cellsmore susceptible to chemotherapy. Based thereon, the present inventionrelates to the use of a cationic liposome preferably comprised of atleast one cationic lipid selected from the group consisting ofdimethyldioctadecyl ammonium bromide (DDAB), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), N-2, S-(dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride,1-[2-(9(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)heptadecenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride; 1,2-(dioleoyl-3-trimethyl ammonium propane (DOTAP), incombination with phosphatidylcholine (PC) and cholesterol (CHOL)containing at least one oligonucleotide to chemosensitize tumor tissueto the effects of chemotherapy. Preferably, the molar ratio of cationiclipid: phosphatidylcholine: cholesterol will range from 0.5 to 2.5:2.0to 4.0:0.5 to 0.5 to 2.5, more preferably 0.7 to 1.5:2.5 to 3.5 and 1.0to 2.0; and most preferably 0.8 to 1.2:3.0 to 3.6 and 1.4 to 1.8.

An especially preferred molar ratio for cationic lipid:phosphatidylcholine: cholesterol is about 1:3.2:1.6. The subjectcationic liposomes can be prepared by known methods. A preferred methodis described in the Examples infra. Essentially, such method comprisesdissolving lipids in a solvent (e.g., chloroform or methanol),evaporation to dryness, hydration at low temperature, addition of anactive agent (e.g., oligonucleotide) in phosphate buffered saline (PBS),vigorous vortexing and sonication, and removal of unencapsulated oligo.These methods have been found to provide for high entrapmentefficiencies (90% or higher). The resultant liposome encapsulated oligoscan be stored, e.g. at 4° C. and used thereafter. For example, in thecase of liposome encapsulated oligos, the liposomes can be stored for atleast about 2 weeks.

The cationic liposomes of the present invention are used to encapsulatea desired oligonucleotide, and optionally another active agent, e.g., apeptide or protein (e.g., antibody, growth factor, cytokine, enzymehormone, receptor or fragment), drug or chemotherapeutic agent,radionuclide, oligosaccharide, fluorophore, or diagnostic agent (such asa radionuclide, detectable enzyme or fluorophore). In the preferredembodiment, the subject cationic liposomes will be used to encapsulatean antisense oligonucleotide that targets a gene specifically expressedby a tumor that is to be treated with chemotherapy. Even more preferablythe antisense oligonucleotide will bind an oncogene such as raf.

As shown in the examples, it has been found that cationic liposomesaccording to the invention provide for high encapsulation efficiencies,protect oligonucleotides from being degraded in plasma, for prolongedperiods (i.e., 8 hours or longer), are effectively delivered to targetcells and enhance intracellular availability of intact oligonucleotides,and very effectively potentate the effects of chemotherapy andradiotherapy.

Also, as shown in the example herein, the subject oligo containing thecationic delivery system potentates the effects of ionizing irradiationand chemotherapy on tumor tissues, including tumors that are resistantto the effects of chemotherapy and radiation. While not wishing to bebound by any particular theory, it is hypothedsized that the subjectcationic delivery system very effectively delivers an oligo to thetargeted site, i.e., a tumor that expresses a gene targeted by theoligo, and that this inhibits the expression of the gene. This in turnis believed to render these cells more susceptible to the effects ofchemotherapy and/or radiation therapy. One hypothesis is that this hasan effect on the cell that makes the cell more susceptible to apoptosis.Preferably, the oligo will comprise modified bases to enhance stabilityin vivo. However, the present invention embraces the use ofoligonucleotides that do or do not comprise modified bases e.g.modifications to the phosphorothioate backbone, such as phosphorothioatemodified oligos, and lipophilic modified oligos (e.g. cholesterol orpoly-L-lysine).

Preferably, the oligonucleotide will be an antisense oligonucleotidecorresponding to an oncogene such as raf, ras, cot, mos, myc, myb,erb-2, or part of a viral gene, and will inhibit expression of a genethat is involved in cancer onset or progression. In preferredembodiments, these liposome-encapsulated oligonucleotides will be usedto treat cancer resistant to chemotherapy, and optionally irradiationtherapy. As noted, the cationic liposome may also contain active agentsother than the oligonucleotide provided that their incorporation doesnot have an adverse effect on the oligo/cationic delivery system. Thisis contemplated as it is well established to treat cancer by a varietyof different therapeutic regimens. Alternatively, if another activeagent is utilized it may be administered in naked form or using adifferent delivery system.

As discovered, the oligo/cationic delivery may be administered prior,concurrent and/or after chemotherapy or radiation.

Also, the subject liposomes may be used to encapsulate peptides, e.g.,haptenic peptides, proteins, antibodies hormones, growth factor andfragments thereof, chemotherapeutic agents, radionuclides,oligosaccharides, lectins, receptors, cytokines or monokines,antineoplastic agents, and other active agents. Examples thereof includeby way of example interleukins, interferons (α,β,γ), colony stimulatingfactors, tumor necrosis factor, methotrexate, cisplatin, doxorubicin,daonorubicin, fibroblast growth factor, and platelet derived growthfactor.

Examples of radionucleotides include by way of example radioactivespecies of yttrium, indium, and iodine.

The amount of active agent that is encapsulated in the subject liposomewill at most be an amount that maintains liposome stability afterencapsulation. In the case of oligonucleotides, the amount ofoligonucleotide will range from a about 0.1 to 1,000 μg oligo/mg oflipid, more preferably about 1 to 100 μg oligo/mg of lipid, and mostpreferably about 10 to 50 μg oligo/mg of lipid.

However, this amount may vary with different active agents. The subjectliposomes will be administered in combination with pharmaceuticallyacceptable carriers such as glucose, and phosphate buffered saline.Also, the liposomes may include preservatives, emulsifiers orsurfactants often used in the formulation of pharmaceuticals.

The subject active agent containing liposomes may be administered bydifferent methods. Systemic and non-systemic methods of administrationare suitable. Such methods include an injection (intramuscular,intraarterial, intraperitoneal, intravenous, intratumoral or othersite-specific injection, intrathecal, inhalatories, oral administration,and topical methods). Preferred methods of administration includeintratumoral and intravenous methods of administration.

The dosage effective amount will depend upon the encapsulated agent, thedisease or condition treated, the patient treated, other therapies, andother known factors. In the case of oligonucleotides a topical dosagewill be one ranging from about 0.1 μg to about 500 μg. Typically, anamount will be administered that results in blood serum concentrationsof oligo or other agents ranging from about 0.1 μg to 1000 μg/ml.

As discussed, it has been surprisingly discovered that oligonucleotides,e.g. antisense oligonucleotides when used as an adjunct to radiotherapyor chemotherapy, potentate the effects of radiotherapy and chemotherapy.For example, it has been shown that antisense oligonucleotides, e.g.that correspond to oncogenes such as raf, can be used to enhance thesensitivity of tumor cells to chemotherapy and radiotherapy, therebyenhancing efficacy. While not wishing to be bound thereby, it istheorized that such oligonucleotides may render tumor cells moresusceptible to lysis or apoptosis (programmed cell death).

Specifically, this has been demonstrated with raf antisenseoligonucleotides. In this embodiment of the invention, an antisenseoligonucleotide corresponding to an oncogene such as raf will preferablybe administered in liposome encapsulated form, prior, concurrent, orshortly after chemotherapy and/or ionizing radiation therapy.

In combination of the invention that includes radiotherapy, irradiationwill be effected by known methods, e.g., by use of a [Mc_(s)]irradiation, or other suitable device for delivering irradiation totumor sites. The amount of irradiation will be an amount sufficient toprovide for tumor regression or remission. As substantiated by theresults, it has been found that the combined use of antisenseoligonucleotide and radiation or chemotherapy has a synergistic effecton tumor remission, especially on tumors resistant to radiation.Therefore, the present invention may enable use of lower dosages ofradiation or chemotherapy than for previous therapies. However, ofcourse, the amount of radiation or chemotherapy will depend upon factorsincluding the condition of the patient, weight, any other therapies,etc. Selection of suitable radiation dosages and therapeutic regimens iswell within the purview of the ordinary skilled artisan.

The size of the administered oligonucleotide will preferably be no morethan 100 nucleotides, more preferably no more than 40 nucleotides, orfrom about 8 to 40 nucleotides and more preferably from about 15 to 40or 15 to 25 nucleotides. The size of the antisense oligonucleotide isone such that upon in vivo administration it results in an antitumoreffect, by disrupting a gene, the expression of which is involved intumor growth, metastasis or apoptosis, or which sensitizes tumor cellsto radiotherapy.

The present invention embraces the use of the subject oligo/cationicliposomal delivery system in conjunction with any chemotherapeutic orchemotherapeutic combination, the efficiency of which is enhancedthereby. Preferably, the oligo/cationic liposomal delivery system willbe used to treat tumors that are resistant to chemotherapy, e.g., as aresult of prolonged administration of a particular chemotherapeutic.

Particularly, as shown in the examples, it has been observed in severaltumor models that the administration of several oligonucleotidescontained in cationic liposomal formulations according to the inventionincreased the efficacy of a number of different chemotherapeutics in amouse tumor model for human prostate cancer and human pancreatic cancer.Enhanced efficacy was shown on the basis of reduced tumor volumes andtumor growth arrest, relative to animals treated with either theliposomal composition or the chemotherapeutic alone and alsoparticularly relative to the control. This was shown for severaldifferent chemotherapeutics, epirubicin, mitoxantrone, cisplatin, Gemzarand Gemcitabine HCl. These results are contained in FIGS. 23–26.

Based on these results, it is anticipated that the subjectoligo-containing cationic liposomal composition can be used as a meansto enhance other chemotherapeutics alone or in combination including byway of example alkylating agents, antimetabolities, apoptosis inducingagents, platinum co-ordination complexes, natural products, hormones,hormone antagonists, receptor agonists and receptor antagonists,anthracenedione, substituted area methylhydrazine derivatives,adrenocortcal suppressants, small molecule inhibitors, peptides,antibodies and antibody fragments, and enzyme inhibitors, such astyrosine kinase inhibitors.

Specific examples of such chemotherapeutics include doxorubicin,daunorubicin, methotrexate, adriamycin, tamoxifen, toremifene,cisplatin, epirubicin, docetaxal, paclitatol, Gemzar, gemcitabicine HCl,mixotantrone, and other known chemotherapeutics useful for treatment ofcancer.

Examples of cancers wherein the claimed combination therapy is usefulinclude solid and non-solid tumors including those that havemetastasized. The therapy can be used for any stage of cancer rangingfrom pre-cancerous lesions to cancer of advanced stages. Specificexamples include prostate cancer, pancreatic cancer, breast cancer, Band T cell leukemias, and lymphomas, bone cancer, head and neck cancer,stomach cancer, bladder cancer, esophageal cancer, lung cancer (e.g.large cell, small cell) ovarian cancer, testicular cancer, myeloma,sarcoma, carcinoma, brain cancer, and others.

In a preferred embodiment the treated cancer will comprise a rafexpressing tumor, such as human pancreatic or human prostate cancer andthe chemotherapeutic will comprise cisplatin, mixotantrone, epirubicin,gemcitabicine, or Gemzar.

The amount of the chemotherapeutic administrated and the regimen will ingeneral be as is conventional for the particular chemotherapeutic whenadministered alone or in conjunction with other chemotherapeutics. Forexample, such dosages may range from about 0.00001 g/kg body weight toabout 1–5 g/kg body weight, dependent upon the particularchemotherapeutic and if it is combined with other therapies. Thechemotherapeutic agent will be administered prior, concurrent or afteradministration of the oligo/cationic liposomal composition according tothe invention. Preferably, the chemotherapeutic will be administeredafter the liposomal composition. It is theorized that the subjectcationic composition renders tumor cells more susceptible to apoptosis.However, the inventors do not want to be bound by their belief.

The cationic liposomal composition and the chemotherapeutic willtypically be administered parenterally, e.g., by subcutaneous,intraperitinal, intravenous, intramuscular, intratumoral, or intrathecalinjection. The composition will general comprise a carrier or excipient,e.g., buffered saline.

Also the chemotherapeutic may be used in conjunction with othertherapies, e.g., radiation, radiommotherapy (RIT), therapeutic enzymes,hormone therapy, and surgery among others discussed above.

EXAMPLE 1

Materials and Methods

Oligodeoxyribonucleotides

Oligodeoxyribonucleotide sequences directed toward the translationinitiation site of human c-raf-1 cDNA were synthesized at Lofstrand LabsLimited (Gaithersburg, Md., USA) using beta-cyanoethyl phosphoramiditechemistry on a Biosearch 8750 DNA synthesizer. The sense (ATG-S (SEQ IDNO:3)) and antisense (ATG-AS (SEQ ID NO:1)) raf ODN sequences were5′-GCAT-CAATGGAGCAC-3′ (SEQ ID NO:3) and 5′-GTG-CTCCATTGATGC-3′ (SEQ IDNO:1), respectively. One terminal base linkage at each end was modifiedto a phosphorothioate group using 3H-1,2-benzo-dithiole-3-1,1,1-dioxideas the sulfurizing agent. Oligos were synthesized at the 15 μm scale andpurified on reverse phase chromatography columns. For quality control, asmall aliquot of each oligo preparation was ³²P-end-labeled andvisualized by polyacrylamide gel electrophoresis (20% acrylamide and 5%bis) followed by densitometric scanning of the labeled products.

For synthesis of the 5′-fluorescein-labeled ATG-AS/S raf ODN (SEQ IDNO:1), (SEQ ID NO:3); the 3′ and 5′ base linkages were modified tophosphorothioate groups as mentioned above. Fluorescein phosphoramidite(1-dimethoxytriyloxy-2-(N-thiourea-(di-O-pivaloyl-fluorescein)-4-aminobutyl-propyl-3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphor-amidite)was coupled to the 5′ ends during the last three synthesis cycles. Thecoupling consisted of the simultaneous addition of and 15-min incubationwith 0.25 ml of a 0.1-M solution of fluorescein amidite in acetonitrileand 0.25 ml of a 0.45-M solution of tetrazole in acetonitrile. Aftersynthesis, the ODNs were deprotected and cleaved from the support in 1.0ml 30% ammonium hydroxide for 24 h at room temperature. Duringdeprotection, the fluorescein labels were modified to the same structureas when prepared using fluorescein isothiocyanate (FITC). Purificationwas performed using standard reverse phase chromatography cartridges.The purified ODNs were eluted from the cartridges in 1.0 ml 20%acetonitrile, dried and resuspended in water.

Preparation of Cationic Liposomes

Cationic lipids, 1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP),1,2-dimeyristoyl-3-trimethyl ammonium propane (DMTAP), anddimethyldioctadecyl ammonium bromide (DDAB) were purchased from AvantiPolar Lipids (Alabaster, Ala., USA). Blank liposomes were prepared usingone of the three cationic lipids, phosphatidylcholine (PC) andcholesterol (CHOL) in a molar ratio of 1:3.2:1.6. LE-ATG-S (SEQ ID NO:3)and LE-ATG-AS raf ODNs (SEQ ID NO:1) were prepared using DDAB, PC andCHOL in a molar ratio of 1:3.2:1.6. Briefly, the lipids dissolved inchloroform or methanol were evaporated to dryness in a round-bottomedflask using a rotatory vacuum evaporator. The dried lipid film washydrated overnight at 4° C. by adding 1 ml of ODN at 1.0 mg/ml inphosphate-buffered saline (PBS). The film was dispersed by vigorousvortexing and the liposome suspension was sonicated for 5 min in a bathtype sonicator (Laboratory Supplies, Hicksville, N.Y., and USA). The ODNto lipid ratio was 30 μg ODN/mg of lipid. The unencapsulated ODN wasremoved by washing the liposomes and centrifugation three times at 25000g for 30 min in PBS. The ODN encapsulation efficiency was determined byscintillation counting of an aliquot of the preparation in which tracesof ³²P-end-labeled ODN were added to an excess of the unlabeled ODN. Theliposome-encapsulated ODNs were stored at 4° C. and used within 2 weeksof preparation. Blank liposomes were prepared exactly as described abovein the absence of ODN.

Cell Culture

SQ-20B tumor cells were established from a laryngeal squamous cellcarcinoma of a patient who had failed a full course of radiationtherapy.⁴³ Tumor cells were grown as monolayers in Dulbecco's modifiedEagle's medium (GIBCO BRL, Grand Island, N.Y., USA) supplemented with20% heat inactivated fetal bovine serum (FBS), 2 mM glutamine, 0.1 mMnonessential amino acids, 0.4 μg/ml hydrocortisone, 100 μg/mlstreptomycin and 100 μg/ml penicillin.

Intracellular raf ODN Uptake and Stability Assays

Logarithmically growing SQ-20B cells were seeded into six-well plates1×10⁶ cells per well) in 20% FBS containing medium. The next day, cellswere switched to 1% FBS containing medium and incubated at 37° C. with10 μM ³²P-labeled LE-ATG-AS raf ODN (SEQ ID NO:1) or ATG-AS raf ODN (SEQID NO:1) (1×10⁶ c.p.m./ml). Following incubation for various intervals,cells were washed with PBS, trypsinized and centrifuged. The cell pelletwas rinsed twice with PBS, resuspended in 0.2-M glycine (pH 2.8) andthen washed again with PBS. This treatment strips off the membrane-boundODN, and the remaining radioactivity was interpreted as representativeof the intracellular level of ODN. The cell pellet was then lysed in 1%SDS and the intracellular radioactivity were determined by liquidscintillation counting.

For ODN stability studies, cells were seeded and incubated with 10 μM³²P-labeled LE-ATG-AS raf ODN (SEQ ID NO:1) or ATG-AS raf ODN (SEQ IDNO:1) (1×10⁶ c.p.m./ml) for 4 h at 37° C. in 1% FBS containing medium.Following this initial incubation with ODN, cells were washed threetimes with PBS and switched to 20% FBS containing medium. Incubationscontinued for various times, followed by trypsinization and washing withPBS. The cell pellets were lysed in 10 mM Tris-HCl, 200 mM NaCl, 1% SDS,200 μg/ml proteinase K, pH 7.4 for 2 n at 37° C. ODNs were extractedwith phenol: chloroform, and the aqueous fractions were collected andaliquots were analyzed by scintillation counting. The samples werenormalized for equal radioactivity in order to correct for a possibleODN efflux over time, followed by electrophoresis in a 15%polyacrylamide/7 M urea gel, and autoradiography.

Pharmacological Disposition Studies of raf ODN

Male Balb/c nu/nu mice (Charles River, Raleigh, N.C., USA; 10–12 weeksold) were maintained in the Research Resources Facility of theGeorgetown University according to accredited procedure, and fed purinachow and water ad libitum. Mice were injected intravenously via the tailvein with 30 mg/kg of LE-ATG-AS raf ODN (SEQ ID NO:1) or ATG-AS raf ODN(SEQ ID NO:1). At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and 48h after injection, one animal in each group was bled from theretro-orbital sinus into heparinized tubes under anesthesia, and killedby cervical dislocation. The blood was centrifuged immediately at 300 gfor 10 min at 4° C. to separate the plasma. The liver, kidneys, spleen,heart and lungs were rapidly excised and rinsed in ice-cold normalsaline. The plasma and organs were stored at −70° C. until furtheranalysis.

Antisense raf ODN (SEQ ID NO:1) was isolated from plasma samples usingthe phenol-chloroform extraction method, and from tissues using a DNAextraction kit (Stratagene, La Jolla, Calif., and USA). The raf ODNconcentration standards were prepared by adding known amounts of ATG-ASraf ODN (SEQ ID NO:1) in blank plasma or blank tissue samples, followedby extraction as mentioned above. The extracts were loaded on to 20%polyacrylamide/3 M urea gels and electrophoresed in TBE buffer. The gelswere electroblotted on to nylon membranes in 0.5×TBE buffer at 20 V for1 h, and the blots were probed with ³²P-labeled sense probe (ATG-S rafODN) (SEQ ID NO:3) in Quickhyb buffer (Stratagene) at 30° C. overnight.The radiolabeled probe was generated by 5′-end-labeling of ATG-S′ rafODN (SEQ ID NO:3) with γ³²-P-ATP using T4 polynucleotide kinase andpurification over Chroma Spin-10 columns (Clontech, Palo Alto, Calif.,and USA). A 10- to 50-fold excess of the probe was used to ensuresaturation of all bands. The autoradiographs were scanned using acomputer program (ImageQuant software, Molecular Dynamics, Sunnyvale,Calif., and USA), and the amounts of ATG-AS raf ODN (SEQ ID NO:1) invarious samples were calculated by comparison to standards.

In Vivo Delivery of S/AS raf ODN (SEQ ID NO:3); (SEQ ID NO:1)

Logarithmically growing SQ-20B cells were injected subcutaneously (2×10⁶cells) in the flank regions on both sides in male Balb/c nu/nu miceunder mild anesthesia. Tumors were allowed to grow to a mean tumorvolume of 115 mm³ before initiation of ODN treatment. Two treatmentroutes were followed: intravenous and intratumoral. For intravenousdelivery, mice were randomly divided into six groups. Three mice in eachgroup received LE-ATG-AS (SEQ ID NO:1), LE-ATG-S (SEQ ID NO:3), ATG-AS(SEQ ID NO:1), ATG-S (SEQ ID NO:3), blank liposomes or normal salineintravenously by bolus infusion via tail vein at a dose of 6 mg/kg dailyfor 5 days. Mice were killed 24 h after the last treatment, and theorgans and tumor tissue were rapidly excised, rinsed in ice-cold normalsaline and stored at −70° C.

For intratumoral delivery, mice were randomly divided into three groups.Three mice in one group received intratumoral injections of 4 mg/kgLE-ATG-AS raf ODN (SEQ ID NO: 1) on the right flank, and LE-ATG-S rafODN (SEQ ID NO:3) on the left flank, daily for 7 days. Two controlgroups, three mice per group, received normal saline or blank liposomes.Mice were killed 24 h after the last treatment, and the tumor tissue wasexcised, rapidly rinsed in ice-cold normal saline and stored at −70° C.

RAF-1 Immunoprecipitation and Immunoblotting Assays

For in vitro experiments, logarithmically growing SQ-20B cells wereexposed to LE-ATG-AS raf ODN (SEQ ID NO:1), LE-ATG-S raf ODN (SEQ IDNO:3) or blank liposomes for various doses and time intervals in 1% FBScontaining medium. Following incubation, cells were lysed in the buffercontaining 500 mM Hepes (pH 7.2), 1% NP-40, 10% glycerol, 5 mM sodiumorthovanadate, 1 mM phenylmethysulfonyl fluoride, 20 μg/ml leupeptin.The lysates were clarified by centrifugation at 16000 g for 20 min andthe protein concentrations were determined (Pierce, Rockford, Ill.,USA). Whole cell lysates, normalized for protein content, were used forimmunoprecipitation of Raf-1 using protein A-agarose conjugated rabbitpolyclonal antibody against 12 carbosy terminal amino acids of humanRaf-1 p74 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA). Theimmunoprecipitates were sequentially washed with cell lysis buffer, 0.5M LiCl 100 mM Tris-HCl, pH 7.4, and 10 mM Tris-HCl, pH 7.4. The immunecomplexes were boiled in Laemmli sample buffer and resolved by 7.5%SDS-PAGE, followed by immunoblotting with polyclonal anti-Raf-1 antibodyand detection of Raf-1 using ECL reagents according to themanufacturer's protocol (Amersham Corporation, Arlington Heights, Ill.,USA). Raf-1 protein expression was quantified using the computersoftware program (Image-Quant; Molecular Dynamics, USA).

For in vivo expression studies, tumor tissue and organs were homogenizedin the cell lysis buffer using Polytron homogenizer (Westbury, N.Y.,USA). Raf-1 expression was analyzed in tissue homogenates byimmunoprecipitation and immunoblotting as described above.

RAF-1 Protein Kinase Activity Assay

Logarithmically growing SQ-20B cells were treated with 10 μM LE-ATG-ASraf ODN (SEQ ID NO:1), LE-ATG-S raf ODN (SEQ ID NO:3) or blank liposomesfor 8 h in 1% FBS containing medium. Cells were lysed as describedabove, and lysates, normalized for protein content, wereimmunoprecipitated with agarose-conjugated anti-Raf-1 antibody overnightat 4° C. Raf-1 phosphotransferase activity was assayed in vitro usingits physiological substrate, mitogen-activated protein kinase (MKK1)³²in kinase buffer containing 30 mM Hepes (pH 7.4), 1 mM manganesechloride, 1 mM DTT, 0.1 mM ATP, and 20 μCi [λ-³²P] ATP (6000 Ci/mmol) asdescribed before.²¹ Radiolabeled reaction products were separated by 10%SDS-PAGE and auto-radiographed. The MKK1 bands were quantified using theImage-Quant program (Molecular Dynamics).

Radiation Survival Dose Response Assay

The appropriate numbers of SQ-20B cells were seeded in duplicate T-25tissue culture flasks (Corning, N.Y., USA) in medium containing 20% FBS,and allowed to attach for 8 h at 37° C. The medium was replaced withmedium containing 1% FBS and the cells were exposed 5 to 10 μM LE-ATG-ASraf ODN (SEQ ID NO:1), 10 μM LE-ATG-S raf ODN (SEQ ID NO:3) or 10 μMblank liposomes for 6 h before irradiation. Irradiations were performedusing ¹³⁷Cs gamma irradiator (JL Shepard MARK I irradiator) and a doserate of 114 cGy/min. The cells were irradiated with total doses of 1 Gy,3 Gy, 5 Gy and 13 Gy, followed by incubation for 2 h. The medium in allflasks was then replaced with 20% FBS containing medium and incubationscontinued for 7–10 days. Surviving colonies were fixed and stained with0.5% methylene blue and 0.13% carbol fuchsin in methanol. Coloniescontaining 50 or more cells were scored and data were fitted to thecomputer-generated single-hit multitarget and linear-quadratic models ofradiation survival response.

EXPERIMENTAL RESULTS

PC/CHOL/DDAB Liposomal Formulation of raf ODN is Nontoxic

Cationic liposomes were prepared using a combination of one of the threecationic lipids (DDAB, DOTAP and DMTAP), phosphatidylcholine (PC), andcholesterol (CHOL) in a molar ratio of 1:3.2:1.5 as described inMaterials and methods. Initially, we determined the ODN entrapmentefficiency of liposomes by scintillation counting of an aliquot of thepreparation in which traces of radiolabeled antisense (ATG-AS (SEQ IDNO:1)) or sense (ATG-S (SEQ ID NO:3)) raf ODN was added to the initialODN, PC/CHOL/DDAB formulation yielded the maximum ODN entrapmentefficiency (>90%, n=10). PC/CHOL/DOTAP and PC/CHOL/DMTAP liposomes werefound to be highly cytotoxic. Therefore, the subsequent experiments wereperformed using the PC/CHOL/DDAB formulation of liposomes.

Fluorescence image analysis using fluorescein-labeled ATG-AS raf ODN(SEQ ID NO:1) was performed to visualize the encapsulation of ODNs inliposomes. A heterogeneous size population of liposomes was obtainedincluding relatively large (FIG. 7) and small liposomes (<2 microns,data not shown). In general, the liposomes showed a tendency to formsmall aggregates, which could be easily dispersed by gentle shaking. TheODN appeared to be distributed in the lipid bilayers and aqueous spaces.We next compared the effects of blank liposomes (BL),liposome-encapsulated antisense (LE-ATG-AS (SEQ ID NO:1)) and sense(LE-ATG-S (SEQ ID NO:3)) raf ODNs on cell survival. Blank liposomes, ata concentration equivalent to 10.0 μM or less of LE-ATG-AS/S raf ODN(SEQ ID NO:1), (SEQ ID NO:3); were non-toxic as determined by theclonogenic survival and trypan blue dye exclusion methods (data notshown). However, blank liposomes showed cytotoxicity at doses higherthan 20 μM and, therefore, doses of 10 μM or less were used for thesubsequent in vitro studies. For in vivo studies, mice wereintravenously (i.v.) treated with a daily dose of 6 mg/kg blankliposomes for 2 weeks, and then monitored for the next 30 days. No signsof weight loss or discomfort were noted, indicating that this liposomalformulation is nontoxic in vivo.

Liposomal Encapsulation Enhances raf ODN Uptake In Vitro and StabilityIn Vitro and In Vivo

We have previously demonstrated that <2% of the free ATG-AS raf ODN (SEQID NO:1) was taken up by SQ-20B cells at 6 hours when exposed to 100 μMconcentration in the presence of low serum (1%), and the maximal uptakewas approximately 4% at 12 hours after treatment. In the present study,we asked whether liposome encapsulation enhances the uptake andstability of ODN in tumor cells. The kinetics of cellular uptake ofLE-ATG-AS raf ODN (SEQ ID NO:1) in the presence of 1% serum is shown inFIG. 8. The intracellular level of ODN increased over time, reaching aplateau 8 hours after incubation. Approximately 13% of the total appliedLE-ATG-AS raf ODN (SEQ ID NO:1) (10 μM) was incorporated into the cells.These results demonstrate that a significant increase in theintracellular level of ODN was achieved when tumor cells were treatedwith a 10-fold lower concentration of LE-ATG-AS raf ODN (SEQ ID NO:1) ascompared with free ATG-AS raf ODN (SEQ ID NO:1).

To examine the intracellular stability of LE-ATG-AS raf ODN (SEQ IDNO:1), ³²P-labeled ODN was recovered at various times following initialtreatment of cells with radiolabeled LE-ATG-AS raf ODN (SEQ ID NO:1) (10μM) for 4 hours. The integrity of the ODN was determined by denaturinggel electrophoresis as described in Materials and methods. Intact rafODN (15-mer) was identified and no degradation was observed up to 24hours (FIG. 8B Igne). In contrast, cells treated with an equimolarconcentration of free ATG-AS raf ODN (SEQ ID NO:1), showed no detectableODN at all time-points (data not shown). In other studies, 15-mer ODNwas intact following incubation of LE-ATG-AS raf ODN (SEQ ID NO:1) for24 hours in medium containing relatively high levels of serum (15% FBS)(data not shown). These results suggest that liposomal encapsulationprotects raf ODN from serum nuclease-induced degradation.

The plasma pharmacokinetics of LE-ATG-AS raf ODN (SEQ ID NO:1) is shownin FIG. 3. Following i.v. administration, the peak plasma concentrationof 6.39 μg/ml was achieved and intact. ODN could be detected up to 24hours. The decrease in plasma concentration of LE-ATG-AS raf ODN (SEQ IDNO:1) followed a biexponential pattern with an initial half-life (t₁/2β)of 24.5 minutes and a terminal half-life t₁/2β of 11.36 h. The areaunder the plasma concentration-time curve for LE-ATG-AS raf ODN (SEQ IDNO:1) was 5.99 μg.h/ml, with total body clearance of 75.94 ml/min/kg andvolume of distribution of 74.67 l/kg. In contrast, intact, free ATG-ASraf ODN (SEQ ID NO:1) was detectable only at 5 min; with a plasmaconcentration of 9.75 μg/ml. These observations are in agreement withthe in vitro observations, and suggest that free ATG-AS raf ODN (SEQ IDNO:1) is rapidly degraded in plasma, whereas LE-ATG-AS raf ODN (SEQ IDNO:1) is in circulation for up to 24 hours.

The tissue distribution of LE-ATG-AS raf ODN (SEQ ID NO:1) is shown inFIG. 4. Intact ODN was detected in all organs examined up to 48 hours(FIG. 4 a). Following the administration of free ATG-AS raf ODN (SEQ IDNO:1); intact ODN was seen only at 5 minutes in various organs anddegradative products (<15-mer) were subsequently found (data not shown).Previous reports of the pharmacokinetic profiles of the fullyphosphorothiotated ODNs (S-oligos), delivered without a carrier, suggestthat liver and kidney are the preferential sites of ODN accumulation.Our data are in agreement with these studies, however, the possibilityremains that liposomal delivery may have facilitated targeting of ODN tocertain tissues, including liver and kidney. The present findingssuggest that raf ODNs, with only the 3′ and 5′ base linkagesphosphorothioated, are rapidly degraded, and that liposome encapsulationis an effective approach for maintaining the ODN stability in variousorgans for at least 48 h.

Liposome-Encapsulated ATG-AS raf ODN (SEQ ID NO:1) Inhibits Raf-AExpression and Activity In Vitro

The time-course experiments revealed that a maximum inhibition of Raf-1protein expression (52.3±5.7%, approximately 74 kDa) occurred 8 hoursafter incubation of cells with 10 μM LE-ATG-AS raf ODN (SEQ ID NO:1)(FIG. 5 a). The inhibitory effect of LE-ATG-AS raf ODN (SEQ ID NO:1)(AS) was seen up to 24 hours (45.6±9.8%). The levels of Raf-1 proteinwere comparable in the control untreated cells (C), blankliposome-treated cells (BL), and LE-ATG-S raf ODN-treated cells (SEQ IDNO:3) (S) (FIG. 5 a), demonstrating that LE-ATG-AS raf ODN (SEQ ID NO:1)specifically inhibited the Raf-1 protein expression in SQ-20B cells.Dose-response studies showed that 35.94±16.8% and 52.3±5.7% inhibitionof Raf-1 expression occurred after treatment of cells for 8 hours with 5μM and 10 μM LE-ATG-AS raf ODN (SEQ ID NO:1), respectively (FIG. 5 b).

We examined the effect of LE-ATG-AS raf ODN (SEQ ID NO:1) on theenzymatic activity of Raf-1 protein kinase using its physiologicalsubstrate, mitogen-activated protein kinase (MKK1).³² Raf-1 proteinkinase activity was comparable in control, untreated cells and LE-ATG-Sraf ODN-treated cells (SEQ ID NO:3) (10 μM, 8 hours). In concurrencewith the level of inhibition of Raf-1 protein expression, the in vitrophosphotransferase activity of Raf-1 protein kinase was inhibited inLE-ATG-AS raf ODN (SEQ ID NO:1) treated cells compared with controlcells (10 μM, 8 hours; 62.6±9.0%) (FIG. 6).

Liposome-Encapsulated ATG-AS raf ODN (SEQ ID NO:1) inhibits Raf-1Expression In Vivo

In Balb/c nu/nu mice, the endogenous levels of Raf-1 expression variedin different normal tissues, and the expression levels were found to bein the descending order of lung>liver>spleen>heart>kidney (data notshown). Interestingly, anti-Raf-1 antibody recognized two protein bands(approximately 74 kDa and approximately 55 kDa) only in kidneys. It isunclear whether the smaller fragment is a proteolytic product of Raf-1in mouse kidney (FIG. 7). The mouse and human c-raf-1 cDNAs share aconserved nucleotide sequence in the translation initiation region(Leszek Woznowski, personal communication). Therefore, we examined theeffect of ATG-AS/S raf ODN (SEQ ID NO:1); (SEQ ID NO:3) on Raf-1expression in normal mouse tissues. No inhibition of Raf-1 expressionwas observed in normal tissues following i.v. administration of freeATG-AS raf ODN (SEQ ID NO:1) (6 mg/kg, daily for 5 days) (data notshown). However, i.v. administration of the LE-ATG-AS raf ODN (SEQ IDNO:1) (6 mg/kg, daily for 5 days), but not LE-ATG-S raf ODN (SEQ IDNO:3), led to a significant inhibition of Raf-1 (approximately 74 kDa)in liver (51.6±17.4%; n=3), and kidneys (42.2±11.0%; n=3) (FIG. 7).LE-ATG-AS raf ODN (SEQ ID NO:1) associated inhibition of Raf-1 did notoccur in heart and lungs (n=3, data not shown). These observations areconsistent with the normal tissue disposition profiles of LE-ATG-AS rafODN (SEQ ID NO:1), showing a relatively higher accumulation of ODN inliver and kidneys compared with heart and lungs (FIG. 4 b). It remainsto be seen whether inhibition of Raf-1 in liver and kidney is associatedwith any appreciable toxicities to these organs.

Surprisingly, i.v. treatment with LE-ATG-AS raf ODN (SEQ ID NO:1)resulted in variable effects on Raf-1 expression in different SQ-20Btumor xenografts, with levels of inhibition ranging from 37.6 to 57.6%compared with LE-ATG-S raf ODN-treated (SEQ ID NO:3) tumor xenografts(n=3) (FIG. 7). We interpret this to be due to differences in tumorvasculature in different xenografts, impeding the delivery of ODN topoorly perfused tumor sites. Intravenous treatment with free ATG-AS/Sraf/ODN (SEQ ID NO:1); (SEQ ID NO:3) LE-ATG-S raf ODN (SEQ ID NO:3) (S),bland liposomes, or normal saline (C) had no effect on Raf-1 expressionin tumor tissue compared with untreated controls. Variations in thelevel of Raf-1 inhibition observed after i.v. treatment prompted us toinvestigate the effect of intratumoral administration of LE-ATG-AS rafODN (SEQ ID NO:1) or LE-ATG-S raf ODN (SEQ ID NO:3) on Raf-1 expression.Results shown in FIG. 8 demonstrate a significant inhibition of Raf-1protein expression in SQ-20B tumor xenografts following intratumoraltreatment with LE-ATG-AS raf ODN (SEQ ID NO:1) compared with LE-ATG-Sraf ODN (SEQ ID NO:3) (60.3±5.4%; 11−3). Taken together, these datademonstrate that LE-ATG-AS raf ODN (SEQ ID NO:1) inhibits Raf-1expression in a sequence-specific manner in vivo.

SQ-20B cells treated with liposome-encapsulated ATG-AS raf ODN (SEQ IDNO:1) are radiosensitive. Radiation survival dose responses of SQ-20Bcells exposed to LE-ATG-AS raf ODN (SEQ ID NO:1), LE-ATG-S raf ODN (SEQID NO:3), or blank liposomes are presented in Table 1. The platingefficiencies of cells treated with LE-ATG-S/AS raf ODN (SEQ ID NO:1);(SEQ ID NO:3) or blank liposomes were comparable (Table 1). Thesingle-hit, multitarget (target model) and the linear quadratic model(LQ) are most commonly used to analyze cellular radiation survival. Thetarget model is based on the parameters D₀ and fl, where D₀ is theinverse of the terminal slope of the survival curve and fl is theextrapolation of this slope to the ordinate. The higher the D_(O) value,the more resistant are cells to radiation-induced cell killing. Anotherparameter, D_(q) is the measure of the shoulder of the survival curve asthe terminal slope line intersects the abscissa. The LQ model has twomajor parameters: α the linear component characterizing the radiationresponse at lower doses; and β, the quadratic component characterizingthe response at higher doses. The higher the value of βthe moresensitive are the cells to radiation. A model-free parameter, D iscalled the mean inactivation dose and represents the area under thesurvival curve plotted on linear coordinates. Clonogenic cell survivaldata were computer-fitted to the single hit multitarget and thelinear-quadratic models of radiation survival response. Significantdecreases observed in the values of radiobiological parameters, D, D₀and D₀ of SQ-20B cells following treatment with LE-ATG-AS raf ODN (SEQID NO:1) suggest a good correlation between the antisensesequence-specific inhibition of Raf-1 protein kinase andradiosensitization. Based on a ratio of the mean inactivation dose, thedose modifying factor (DMF) of LE-ATG-AS raf ODN (SEQ ID NO:1) treatment(10 μm) was approximately 1.6. These data are significant because a10-fold higher concentration of the free ATG-AS raf ODN (SEQ ID NO:1) isrequired to achieve a comparable level of the radiosensitization ofSQ-20B cells (ATG-AS raf ODN (SEQ ID NO:1), 100 μm; DMF approximately1.4).

TABLE 1 RADIATION SURVIVAL PARAMETERS OF SQ-20B CELLS TREATED WITHLE-ATG-S/AS RAF ODN (SEQ ID NO:3); (SEQ ID NO:1) No. of raf ODNexperiments D₀ (Gy) D₄ (Gy) η α (Gy^(ml)) β (Gy⁻²) D (Gy) Blank 5 2.795± 1.445 ± 2.012 ± 0.2184 ± 0.0087 ± 3.659 ± liposomes/ 0.38 1.22 1.340.11 0.00 0.02 LE-ATG-S (SEQ ID NO:3) LE-ATG-AS 3 2.287 ± 0.051 ± 1.021± 0.4385 ± 0.0000 ± 2.280 ± (SEQ ID 0.23 0.05 0.19 0.05 0.00 0.00 NO:1)

The appropriate numbers of cells were seeded in duplicate T-25 flasksper dose in each experiment as explained in Materials and method.Plating efficiencies of the blank liposome treated, LE-ATG S raf ODN(SEQ ID NO:3) treated and LE-ATG-AS raf ODN (SEQ ID NO:1) treated cellswere in the range of 65–79%, 52–83% and 59–90% respectively.

Composite value of the various parameters were obtained from the threeexperiments performed with LE-ATG raf ODN-treated cells and twoexperiments performed with the blank liposome-treated cells.

ANALYSIS

The above results indicated that a cationic liposome formulationaccording to the invention (PC/CHOL/DDAB) has several advantages.Specifically, the PC/CHOL/DDAB liposomal formulation was found to benontoxic, and yielded a high ODN encapsulation efficiency.

We have identified several in vivo parameters that indicate that thesecationic liposomes are a suitable vehicle to transport antisense oligossafely and effectively. Based on fluorescence microscopy, it appearsthat oligos may be entrapped inside the lipid bilayer (FIG. 1). Theobservations extend the initial reports that showed encapsulation ofplasmid DNA within lipid sheets or tubes. By simultaneously measuringplasma and tissue levels, we also demonstrate that liposomalencapsulation of oligos protects these relatively small pieces of DNAfrom degradation in plasma and facilitates their tissue accumulation(FIGS. 3 and 4). Circulating antisense raf oligos carried in vivo byliposomes were intact for at least 24 hours, while free oligos wereundetectable after five minutes. These data are in agreement withprevious reports showing that phosphodiester oligos with only twoterminal phosphorothioate linkages at the 3′ and 5′ ends resemble theunblocked phosphodiester oligos, and that these oligos are rapidlycleared from the blood and show little tissue accumulation. It ishypothesized that the use of PC along with cholesterol in our liposomalpreparation may have facilitated the prolonged retention of oligos inthe circulation, as well as tissue disposition and stability of oligos.

Particle size has been shown to play a major role in liposomebiodistribution and the route of cell entry. Larger liposomes aredistributed primarily to the reticuloendothelial system with negligibleamounts in other tissues, whereas smaller liposomes are localized toother organs. Additionally, the clearance of multilamellar vesicles ofheterogenous size distribution follows a biphasic pattern, with rapidclearance of larger liposomes and a slow rate of clearance of smallliposomes. Limited information is available on the biodistribution ofcationic liposomes containing oligonucleotides. Litzinger and colleaguespreviously reported that oligonucleotides complexed with cationicliposomes, approximately 2.0 microns in diameter, are transiently takenup by the lungs followed by rapid distribution to liver. Recent studiesdemonstrated that endocytosis is the principal pathway for delivery ofoligonucleotides via cationic liposomes. Our liposomal preparationsconsisted of both large and small liposomes. Consistent with the abovenotion, we demonstrate that the clearance of LE-ATG-AS raf ODN (SEQ IDNO:1) followed a biphasic pattern with preferential distribution toliver.

Liposome-encapsulated antisense raf oligos were non-toxic, and inhibitedRaf-1 expression in vitro and in vivo in a sequence-specific manner(FIGS. 5–8 and Table 1). It is noteworthy that intravenous andintratumoral routes of LE-ATG-AS raf ODN (SEQ ID NO:1) administrationled to a significant inhibition of Raf-1 expression in SQ-20B tumortissue, suggesting the potential applicability of this compound for bothsystemic and local administrations. Furthermore, tumor cells treatedwith liposomal-encapsulated antisense raf oligo were radiosensitivecompared with control cells (Table 1). More recently, in collaborationwith Dr. Brett Monia (ISIS Pharmaceuticals, Carlsbad, Calif., USA),experiments have been initiated to demonstrate the radiosensitizingeffect of liposome-encapsulated antisense raf oligo (5132 (SEQ ID NO:4)) in the SQ-20B tumor xenograft model. The in vivo data obtained sofar in athymic mice are promising (Gokhale et al. unpublished data). Thepresent results suggest that liposomal delivery of ATG-AS raf ODN (SEQID NO:1) in combination with radiation may be an effectivegene-targeting approach for treatment of cancers, especially those thatare resistant to standard radiation therapy.

EXAMPLE 2

Materials and Methods

Cell Culture

SQ-20B tumor cells were grown as a monolayer in Dulbecco's modifiedEagle's medium (DMEM) (GIBCO BRL, Grand Island, N.Y.) supplemented with20% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 0.1 mMnonessential amino acids, 0.4 μg/ml hydrocortisone, 100 μg/mlstreptomycin, and 100 U/ml penicillin.

Oligodeoxyribonucleotides

A 20-mer phosphorothioate antisense ODN (ISIS 5132/5132:5′-TCC-CGC-CTG-TGA-CAT-GCA-TT-3′) (SEQ ID NO:4) corresponding to the3′-untranslated region (3′-UTR) of human c-raf-1 mRNA and a seven-basemismatched phosphorothioate antisense ODN (ISIS 10353/10353;5′-TCC-CGC-GCA-CTT-GAT-GCA-TT-3′ (SEQ ID NO:5)) were designed andsynthesized as described previously (Monia et al., 1996a,b). A 20-merphosphorothioate sense ODN (5′-ATT-GCA-TGT-CAC-AGG-CGG-GA-3′ (SEQ IDNO:6)) was synthesized at Lofstrand Labs Limited (Gaithersburg, Md.) asdescribed previously (Soldatenkov et al., 1997).

Preparation of Cationic Liposomes

ODN was encapsulated in cationic liposomes prepared usingdimethyldioctadecyl ammonium bromide, phosphatidylcholine, andcholesterol (Avanti Polar Lipids, Alabaster, Ala.) in a molar ratio of1:3,2:1,6 as described in previously (Gokhale et al., 1997). Briefly,the lipids dissolved in chloroform or methanol were evaporated todryness in a round-bottomed flask using a rotary vacuum evaporator. Thedried lipid film was hydrated overnight at 4° C. by adding 5132 (SEQ IDNO:4)/10353 (SEQ ID NO:5) at 1.0 mg/ml in phosphate-buffered saline(PBS). The film was dispersed by vigorous vortexing, and the liposomesuspension was sonicated for 5 minutes in a bath-type sonicator(Laboratory Supplies, Hicksville, N.Y.). The ODN/lipid ratio was 30 μgODN/mg lipid, resulting in greater than 90% encapsulation efficiency.The liposome-encapsulated ODN (LE-5132 (SEQ ID NO:4) LE-10353 (SEQ IDNO:5)) was stored at 4° C. and used within a week. Blank liposomes (BL)were prepared exactly as described in the absence of ODN.

Raf-1 Immunoprecipitation and Immunoblotting Assays

For in vitro expression studies, on day 1, logarithmically growingSQ-20B cells were exposed to various concentrations of LE-5132 (SEQ IDNO:4), 5132 (SEQ ID NO:4), LE-10353 (SEQ ID NO:5), or BL in 1%FBS-containing medium for 6 hours. The cells were then washed with 20%FBS containing medium to remove liposomes, and incubation in 20%FBS-containing medium continued overnight (18 hours) in the presence of5132 (SEQ ID NO:4) in the LE-5132 (SEQ ID NO:4) and 5132 (SEQ ID NO:4)treatment groups and the presence of 10353 (SEQ ID NO:5) in the LE-10353(SEQ ID NO:5) group. On day two, cells were rinsed with fresh 30% FBS,followed by a second course of the treatment schedule as on day one foran additional 24 hours. This procedure yielded a minimal exposure ofcells of LE-5132 (SEQ ID NO:4) 12 hours). Cells were then lysed in thebuffer containing 500 mM HEPES, pH 7.2 1% NP-40, 10% glycerol, 5 mMsodium orthovanadate, 1 μM phenylmethysulfonyl fluoride, 20 μg/mlaprotinin, and 20 μg/ml leupeptin. The lysates were clarified bycentrifugation at 16,000 g for 20 minutes, and the proteinconcentrations were determined (Pierce, Rockford, Ill.). Whole celllysates, normalized for protein content, were used forimmunoprecipitation of Raf-1, using protein A-agarose-conjugated rabbitpolyclonal antibody against 12 carboxy-terminal amino acids of humanRaf-1 p74 (Santa Cruz Biotechnology, Santa Cruz, Calif.). Theimmunoprecipitates were sequentially washed with cell lysis buffer, 0.5M LiCl, 100 mM Tris-HCl, pH 7.4, and 10 mM Tris-HCl, pH 7.4. The immunecomplexes were boiled in Laemmli sample buffer and resolved by 7.5%SDS-PAGE. This was followed by immunoblotting with polyclonal anti-Raf-1antibody and detection of Raf-1 using ECL reagents according to themanufacturer's protocol (Amersham Corporation, Arlington Heights, Ill.).The Raf-1 protein level was quantified using the computer softwareprogram Image-Quant (Molecular Dynamics, Sunnyvale, Calif.).

For in vivo expression studies, tumor tissue was homogenized in the celllysis buffer using a Polytron homogenizer (Westbury, N.Y.). Raf-1expression was analyzed in tissue homogenates by immunoprecipitatibn andimmunoblotting as described above.

In Vitro Coagulation Assay

The coagulation, assay was performed using normal human plasmacontaining a known concentration of LE-5132 (SEQ ID NO:4) or 5132 (SEQID NO:4). The activated partial thromboplastin time (APTT) was measuredafter adding APTT reagent (rabbit brain cephalin extract with ollagicacid activator) (Sigma Diagnostics, St. Louis, Mo.) to plasma samples,followed by addition of calcium chloride to initiate clot formationaccording to the manufacturer's recommendations (Sigma Diagnostics). Thetime required to form visible clots was recorded manually.

Pharmacokinetic Studies

Male Balb/c nu/nu mice (National Cancer Institute, Frederick, Md.) weremaintained in the Division of Comparative Medicine, GeorgetownUniversity, according to accredited procedures, and fed purina chow andwater ad libitum. Mice were injected i.v. via the tail vein with 30mg/kg of LE-5132 (SEQ ID NO:4) or 5132 (SEQ ID NO:4) formulated in PBS.At 5, 15, and 30 minutes and 1, 2, 4, 8, 24, and 48 hours afterinjection, animals were bled, under anesthesia, from the retroorbitalsinus into heparinized tubes and killed by cervical dislocation. Theblood was centrifuged immediately at 300 g for 10 minutes at 4° C. toseparate the plasma. The liver, spleen, kidney, heart, and lungs wererapidly excised and rinsed in ice-cold normal saline. The plasma andorgans were stored at −70° C. until further analysis.

Antisense raf ODN concentrations in plasma and tissue samples weredetected as we described earlier (Gokhale et al., 1997). Briefly, theODN was isolated from plasma samples using the phenol-chloroformextraction method and from tissues using a DNA extraction kit(Stratagene, La Jolla, Calif.). The raf ODN concentration standards wereprepared by adding known amounts of 5132 (SEQ ID NO:4) in blank plasmaor blank tissue samples, followed by extraction as described earlier.The extracts were loaded onto 20% pblyacrylamide/8 M urea gels andelectrophoresed in TBE buffer. The gels were electroblotted onto nylonmembranes in 0.5×TBE buffer at 20 V for one hour, and the blots wereprobed with [³²P]-labeled sense raf ODN probe in Quickhyb buffer(Stratagene) at 30° C. overnight. A 10–50-fold excess of the probe wasused to ensure saturation of all bands. The autoradiographs were scannedusing a computer program (Image-Quant software), and the amounts ofantisense raf ODN in the samples were calculated by comparison withstandards.

In Vivo LE-5132 (SEQ ID NO:4) Treatment and Irradiation Procedures:Antitumor Efficacy Study Design

Logarithmically growing SQ-20B cells were injected s.c. (2×10^(o) cells)in the left flank region in male Balb/c nu/nu mice under mildanesthesia. Tumors were allowed to grow to a mean tumor volume of ˜72–94mm³ before initiation of treatment. Volumes for tumors were determinedfrom caliper measurements of the three major axes (a, b, c) andcalculated using abc/2, an approximation for the volume of an ellipse(πabc/6).

For each experiment, tumor-bearing mice were randomly divided intodifferent treatment group, with 5–7 animals per group. The ODN group ofmice received LE-5132 (SEQ ID NO:4) i.v. at a dose of mg/kg daily or onalternate days for a total of 12–18 days or by both methods. The tumorsin the IR group were irradiated using a [¹³⁷Cs] irradiator (JJ. ShepardMark I). Animal restraint and shielding of normal tissues wereaccomplished within a hinged hemicylindrical plastic chamber mountedbehind a specially shaped 2.5 cm thick lead shield. The tumor-bearinghind limb protruded through a hole in the chamber and was mounted, bytaping the foot, on a 1.6 mm Plexiglas platform exposed to theirradiation. Using TLD dosimetry, the average dose rate to the center ofthe tumor and to the mouse body were determined beforehand using theexperimental irradiation conditions and a custom-made,tissue-equivalent, mouse phantom. Dose distributions were confirmed onseveral mice. Radiation was delivered to the tumors at a dose rate of2.37 Gy/min. for 3.8 Gy daily for up to 18 days. The whole body dose was<5% of the tumor dose (0.19 Gy/day). No gastrointestinal problems werenoted for the duration of the experiment in any mice. The combinedtreatment group of animals received LE-5132 (SEQ ID NO:4) on day 0 andLE-5132 (SEQ ID NO:4) and IR at an interval of 3–4 hours on days 1–6,and on days 8, 10, and 12. In addition, this group received IR alone ondays 7, 9, and 11 and days 13–18. Of the two control groups, onereceived BL on the same dosing schedule as LE-3132 (SEQ ID NO:4) (BL)and the other was left untreated (C). Tumor volumes were monitored onceor twice weekly and for 12–27 days after the final treatment.

Tumor volumes were calculated as the percentage of initial tumor volume(day 0, the first day of dosing), and mean tumor volume±SE was plotted.Analysis of variance (one-way ANOVA) was performed to determine thestatistical significance of changes in tumor volumes observed on day 12after the final treatment.

Histopathology

Tumor tissues were excised from representative treatment groups 24 hoursafter the last treatment and fixed in 10% formalin, and paraffinsections were analyzed microscopically after hematoxylin/cosin staining.Histologic changes, such as apoptotic cells containing fragmentedchromatin, were scored under the light microscope (American OpticalCorporation, Buffalo, N.Y.). Ten fields with approximately 100 cells perfield were scored in each treatment group.

EXPERIMENTAL RESULTS

LE-5132 (SEQ ID NO:4) Inhibits Raf-1 Expression In Vitro

To establish the antisense ODN sequence-specific inhibition of Raf-1expression, SQ-20B tumor cells was treated with 5132 (SEQ ID NO:4),LE-5132 (SEQ ID NO:4), and a seven base mismatch LE-10353 (SEQ ID NO:5)(FIG. 9A). Previously, in vitro inhibition of Raf-1 with 5132 (SEQ IDNO:4) has been shown to require lipofectin (Monia et al., 1996a).Consistently, 5132 (SEQ ID NO:4) was found to be ineffective in cellculture (FIG. 9A, lanes 4 and 6). A significant decline in the level ofRaf-1 protein was observed with LE-5132 (SEQ ID NO:4) compared with 5132(SEQ ID NO:4) treatment in vitro (FIG. 9B, 0.5 μM LE-5132 (SEQ ID NO:4),71.3±22.5%; 1.0 μM LE-5132 (SEQ ID NO:4), 79.6%±16.7%).Liposome-encapsulated mismatch antisense ODN (LE-10353) (SEQ ID NO:5)showed Raf-1 expression comparable to untreated or BL-treated cells(FIG. 9A, lanes 7–9). Taken together, these data establish the antisensesequence specific potency of LE-5132 (SEQ ID NO:4) in SQ-20B cells.

LE-5132(SEQ ID NO:4) Does not Affect Coagulation In Vitro

As a step toward the clinical application of cationic liposomes todeliver ODN safely and effectively, we compared the effects of 5132 (SEQID NO:4) and LE-5132 (SEQ ID NO:4) on coagulation time, using normalhuman plasma. Addition of 5132 (SEQ ID NO:4) to normal human plasmaproduced concentration-dependent prolongation of clotting time (FIG.10). Approximately 95% and 197.5% increases in APTT were observed invitro in the presence of 100 μg/ml and 200 μg/ml of 5132 (SEQ ID NO:4),respectively, whereas only marginal increases in APTT were seen withLE-5132 (SEQ ID NO:4) (100 μg/ml, 13%; 200 μg/l, 14.5%). BL in the sameconcentration range showed no effect on APTT (data not shown).

Liposome-Encapsulation Enhances Pharmacokinetics of 5132 (SEQ ID NO:4)

The pharmacokinetic parameters were obtained after a single i.v. bolusadministration of LE-5132 (SEQ ID NO:4) or 5132 (SEQ ID NO:4). As shownin FIGS. 11A and B, intact ODN could be detected in plasma for at leastup to 8 hours in both cases. The peak plasma concentrations at 5 minutesafter ODN administrations were 28.5 μg/ml and 13.5 μg/ml for LE-5132(SEQ ID NO:4) and 5132 (SEQ ID NO:4), respectively. The decrease inplasma concentration of LE-5132 (SEQ ID NO:4) and 5132 (SEQ ID NO:4)followed a biexponential pattern with initial distribution half-life(t₁/2β) of 34 minutes and 21.6 minutes, respectively. The terminalhalf-lives (t₁/2β) with LE-5132 (SEQ ID NO:4) and 5132 (SEQ ID NO:4)were 14.5 hours and 4.3 hours, respectively. As shown in Table 2, thearea under the plasma concentration-time curve (AUC) was 5.8 timeshigher with LE-5132 (SEQ ID NO:4) compared with 5132 (SEQ ID NO:4), andthe rate of clearance of intact ODN was higher with 5132 (SEQ ID NO:4)compared with LE-5132 (SEQ ID NO:4).

The normal tissue distribution profiles of LE-5132 (SEQ ID NO:4) and5132 (SEQ ID NO:4) are presented as a function of the AUC in FIG. 12.Following either treatment, intact ODN could be detected up to 48 hoursin the organs examined (data not shown). However, the tissuedistribution of LE-5132 (SEQ ID NO:4) was different from that of freephosphorothioated ODN, 5132 (SEQ ID NO:4). Significantly higher levelsof intact ODN could be measured in liver (18.4-fold) and spleen(31-fold) after LE-5132 (SEQ ID NO:4) administration compared with 5132(SEQ ID NO:4) administration. Slightly higher ODN levels were noticed inother organs via liposomal delivery of ODN compared with free ODN(heart, 3-fold; lungs, 1.5-fold). Interestingly, the level of intact ODNin kidneys was lower with LE-5132 (SEQ ID NO:4) (0.77-fold). Additionalstudies performed indicated a modestly higher ODN level in SQ-20B tumortissue following LE-5132 (SEQ ID NO:4) treatment compared with 5132 (SEQID NO:4) treatment (1.4-fold).

LE-5132 (SEQ ID NO:4) Inhibits SQ-20B Tumor Growth

As shown in FIG. 13, antitumor effects of LE-5132 (SEQ ID NO:4) wereobserved within a week after treatment initiation, and mean tumor volumecontinued to decrease during the course of subsequent treatments. On thefinal day of treatment (day 19), mean tumor volumes were 42.0%+5.5% and290.6%+26.6% of initial volume (day 0, 100%) in the LE-5132 (SEQ IDNO:4) and BL groups, respectively. The tumor volume in the LE-5132 (SEQID NO:4) group reached the initial volume within 6–10 days after thelast dosing. A remarkable difference in the tumor volumes was noticed inthe LE-5132 (SEQ ID NO:4) and BL groups at all times after treatment.The study was terminated on day 30, at which time the mean tumor volumein the BL group was approximately 3.4-fold more than that of the LE-5132(SEQ ID NO:4) group.

In other studies, we compared the antitumor efficacies of LE-5132 (SEQID NO:4) and 5132 (SEQ ID NO:4). LE-5132 (SEQ ID NO:4) or 5132 (SEQ IDNO:4) (10 mg/kg) was administered i.v. into tumor-bearing mice daily forthe first 7 days, followed by three additional doses on alternate days.The control group received similar treatment with BL or was not treatedat all (C). Tumor volumes were monitored for a total of 35 days. Meantumor volumes on day 35 compared with day 0 (100%) were: BL,427.8%±32.5%; C, 405.3%±26.8%; 5132 (SEQ ID NO:4), 159.6%±10.0%; LE-5132(SEQ ID NO:4), 105.3%±6.3%. ANOVA was performed to determine thesignificance of difference in mean tumor volumes in various categorieson day 35. Tumor growth patterns were comparable in the BL and C controlgroups. Both the 5132 (SEQ ID NO:4) and LE-5132 (SEQ ID NO:4) groupsdisplayed significant antitumor activity vs. the BL and C groups (n=5,p<0.0001). However, the LE-5132 (SEQ ID NO:4) group displayed greaterantitumor activity relative to 5132 (SEQ ID NO:4) (n=5, p<0.001). Thesedata are consistent with relatively increased plasma, normal tissue, andtumor levels of 5132 (SEQ ID NO:4) in the liposome-encapsulated form.

LE-5132 (SEQ ID NO:4) Inhibits Raf-1 Expression In Vivo

Relative Raf-1 protein levels were measured in SQ-20B tumor tissue inmice exposed to LE-5132 (SEQ ID NO:4)±IR or BL (FIG. 14).

TABLE 2 EFFECT OF LBPOSOME ENCAPSULATION ON PHARMACOKINETIC PARAMETERSOF ODN^(a) t₁/2_(β) ^(b) C_(max) AUC_(area) CL Vd_(area) ODN (hours)(μg/ml) (μg · h/ml) (L/h/kg) (L/kg) 5132 4.30 13.57 6.20 4.82 29.96 (SEQID NO:4) LE-5132 14.50 28.50 36.60 0.82 17.15 SEQ ID nO:4) ^(a)30 mg/kgbolus, i.v. in Balb/c nu/nu mice. ^(b)t₁/2_(β), elimination half-life;C_(max). peak plasma concentration; AUC, area under the plasmaconcentration-time curve; CL, total body clearance; Vd_(area), volume ofdistribution.

In the LE-5132 (SEQ ID NO:4) group, Raf-1 expression was found to be35.5%±13.4% and 27.7%±13.3% compared with the BL group (100%) on day 7and day 14, respectively. Inhibition of Raf-1 expression was also notedin SQ-20B tumors in mice treated with a combination of LE-5132 (SEQ IDNO:4) and IR. IR treatment alone did not change Raf-1 expressioncompared with the untreated or BL control (FIG. 14).

LE-5132(SEQ ID NO:4) is a Tumor Radiosensitizer

Because SQ-20B cells were established from a tumor after failure ofradiation therapy and LE-5132 (SEQ ID NO:4) treatment caused tumorgrowth arrest during the course of treatment, we asked if control ofgrowth of this relatively radioresistant tumor could be achieved by acombination of LE-5132 (SEQ ID NO:4) and IR treatments. LE-5132 (SEQ IDNO:4) (10 mg/kg) was administered ten times over 12 days (day 0–day 12).This treatment caused tumor growth arrest compared with control groupsof mice (untreated and BL) for up to one week after the last dosing (day19). This was followed by a steady increase in tumor volume (FIG. 15 A).IR (3.8 Gy/day) was given daily for eighteen days. In this group, amodest decline in mean tumor volume was observed by day 26 (14.9%+7.1%of initial volume) (FIG. 7A). Tumors grew thereafter and reached theinitial volume within the next in 3–7 days. The combination of LE-5132(SEQ ID NO:4) and IR treatment caused a significant decrease in the meantumor volume by day 26 (57.9%±8.0% of initial volume). By day thirty,the mean tumor volumes compared with initial volumes (100%, day 0) were:LE-5132 (SEQ ID NO:4), 122.5%±13.8%; IR, 113.8%±17.6%; LE-5132 (SEQ IDNO:4)±IR, 43.5%±2.9%; LB/untreated control, 370.8%±15.6% (FIG. 15). BothLE-5132 (SEQ ID NO:4) and IR groups displayed significant tumor growtharrest vs. BL and untreated groups (p<0.001). Statistical analysisindicated that tumor volume difference was insignificant in the IR vs.the LE-5132 (SEQ ID NO: 4) group. Most important, the LE-5132 (SEQ IDNO:4)+IR group displayed significantly greater antimmor activity vs.LE-5132 (SEQ ID NO:4), IR, BL, and untreated groups (p<0.001) FIG. 15B)

Combination of LE-5132 (SEQ ID NO:4) and IR Treatments CausesSignificant Increase in Apoptosis In Vivo

Representative tumors in various treatment groups were excised 24 hoursafter the last treatment for histopathologic examination. Both necroticand apoptotic cells were seen in the LE-5132 (SEQ ID NO:4) or IR groupcompared with the untreated group. In addition, clonal regrowth of someviable cells containing intact nuclei was observed in the IR group (datanot shown). The proportion of apoptotic cells containing highlyfragmented nuclei was considerable higher in the LE-5132 (SEQ IDNO:4)+IR group compared with the single agent or untreated control group(FIG. 16). The ratios of the relative number of apoptotic cells/viablecells scored in different groups were: C, 0.06 LE-5132 (SEQ ID NO: 4),0.64; IR, 0.72; LE-5132 (SEQ ID NO:4)+IR, 2.46.

DISCUSSION

Antisense ODN therapeutics is a novel approach to enhance the efficacyof an anticancer agent via sequence-specific inhibition of aproliferative or survival signal. To our knowledge, this report providesthe first evidence of the effectiveness of a well-characterizedantisense ODN as a radiosensitizer or chemosensitizer in an animal tumormodel. SQ-20B tumor cells were established from a laryngeal squamouscell carcinoma of a patient who had failed a full course of radiationtherapy. Radiation or antisense raf ODN treatment alone caused temporaryinhibition of SQ-20B tumor growth but not tumor regression, whereas acombination of antisense raf ODN and radiation treatments led tosustained tumor regression for at least 27 days after treatment (FIGS.13 and 15). These data support the role of Raf-1 in cell proliferationand survival and establish antisense rad ODN as a novel in vivoradiosensitizer.

We found significant inhibition of Raf-1 protein expression followingLE-5132 (SEQ ID NO:4) treatment of SQ-20B cells and tumor, suggestingthat LE-5132 (SEQ ID NO:4) is a biologically active compound in vitroand in vivo (FIGS. 9 and 14). Because the 5132 (SEQ ID NO:4) sequencecorresponds to a 3′ UTR of human c-raf-1 not conserved in mouse,inhibition of Raf-1 in normal mouse tissues could not be investigated.Previous studies have indicated that among other potentially toxiceffects, PS-ODN treatment causes bruising associated with dose-dependentprolongation of the clotting time. Complement and coagulation effects ofPS-ODN including 5132 (SEQ ID NO:4) could be avoided by altering thedosing regimen. Our results show that liposomal encapsulation of 5132(LE-5132 (SEQ ID NO:4)) prevents changes in coagulation time (FIG. 10).Also, liposomal delivery of ODN may alleviate many of the othersequence-independent side effects of PS-ODN, including hematologicchanges and complement activation. Significant elevation in plasmaconcentration and most tissue levels of the liposomal formulation of5132 (SEQ ID NO:4) was observed compared with free 5132 (SEQ ID NO:4)(FIGS. 11 and 12 and Table 2). Consistent with this, antitumor potencyof LE-5132 (SEQ ID NO:4) was found to be significantly higher than thatof 5132 (SEQ ID NO:4). Taken together, these data suggest that liposomeencapsulation is an efficacious method of ODN transport in vivo.

The mechanism by which inhibition of Raf-1 expression enhancesIR-induced cytotoxicity is not clear. The role of Raf-1 as anantiapoptotic or survival factor has been demonstrated in growthfactor-deprived hematopoietic cells and in v-abl-transformed NIH/3T3cells (Troppmair et al., 1992; Wang et al., 1996, Weissinger et al.,1997). Several reports indicate that a balance between cell death andcell survival signals determines the fate of the cells exposed togenotoxic or nongenotoxic stress. IR has been shown to activate diversetypes of signaling molecules, including Raf-1 protein kinase,mitogen-activated protein kinase (MARK), and transcription factors AP-1and NK-/κB (reviewed in Kasid and Suy, 1998). One possibility is thatRaf-1 may have a protective role in irradiated cells. We have observedincreased level of Bax protein, a proapoptotic member of the Bcl-2family, in SQ-20B cells treated with either IR. or a combination ofLE-5132 (SEQ ID NO:4) and IR (data not shown). Radiation-inducible Baxexpression has been correlated with apoptosis (Zhan et al., 1994).Furthermore, histopathologic examination revealed a significantproportion of tumor cells containing fragmented chromatin, indicative ofapoptosis in the LE-5132 (SEQ ID NO:4)+IR treatment group compared withthe LE-5132 (SEQ ID NO:4), IR, or untreated control groups (FIG. 16).Those data suggest that Raf-1 may serve to promote the antiapoptoticsignaling pathway(s) in irradiated cells. Inhibition of Raf-1 withantisense raf ODN would then result in the substantial effects ofIR-responsive proapoptotic signals, including reversal of tumorradioresistance.

EXAMPLE 3

Materials and Methods

Preparation of DMTAP:PC:CHOL Liposomes

Liposomes having a molar ratio of 1,2-dimyristoyl-3-trimethyl ammoniumpropane (DMTAP): phosphatidylcholine (PC): and cholesterol (CHOL), of1:3.2:1.6, and having encapsulated therein an antitumor rafoligonucleotide (ATG-AS (SEQ ID NO:1)) were prepared using substantiallythe same methods described previously.

In Vitro Results

A Enhanced Cellular Uptake of Antisense raf OligodeoxyribonucleotidesEncapsulated in Liposomes Comprised of DMTAP:PC:CHOL.

Dose-response uptake experiments: SQ-20B tumor cells were incubated witha mixture of radiolabeled (³²P-γATP) and an indicated dose of unlabeledantisense raf oligonucleotide (ATG-AS (SEQ ID NO:1)) either in theliposome encapsulated form (LE-ATG-AS (SEQ ID NO:1)) or free form(ATG-AS (SEQ ID NO:1)) (FIG. 17). The treatment lasted for 4 hours at37° C. in 1% serum containing medium. Following incubation, cells werewashed with phosphate buffered saline (PBS), detached by trypsinization,and collected by centrifugation. The cell pellet was washed twice withPBS, and cells were then lysed in 1% sodium dodecyl sulphate. Theintracellular radioactivity indicative of the amount of ATG-AS (SEQ IDNO:1) taken up by the cells was determined by liquid scintillationcounting. Data shown in FIG. 17 indicate a significant increase in theintracellular uptake of LE-ATG-AS (SEQ ID NO:1) at all doses testedcompared to ATG-AS (SEQ ID NO:1) (1, 2, 4, 8 and 10 μM).

Time-course uptake experiments: SQ-20B tumor cells were incubated with amixture of radiolabeled (³²P-γATP) and 4 μM of unlabeled anti sense rafoligonucleotide (ATG-AS (SEQ ID NO:1)) either in the liposomeencapsulated form (LE-ATG-AS (SEQ ID NO:1)) or free form (ATG-AS (SEQ IDNO:1)). The treatment lasted for indicated times at 37° C. in 1% serumcontaining medium (FIG. 18). Following incubation, 15 cells were washedwith phosphate buffered saline (PBS), detached by trypsinization, andcollected by centrifugation. The cell pellet was washed twice with PBS,and cells were then lysed in 1% sodium dodecyl sulphate. Theintracellular radioactivity indicative of the amount of ATG-AS (SEQ IDNO:1) taken up by the cells was determined by liquid scintillationcounting. Data shown in FIG. 18 indicate a significant increase in theintracellular accumulation of LE-ATG-AS (SEQ ID NO:1) at all time pointstested compared to ATG-AS (SEQ ID NO:1) (15 minutes, 30 minutes, 1 hour,2 hours, 4 hours, 6 hours, 16 hours, and 24 hours).

B. Intracellular Stability of Antisense raf OligodeoxyribonucleotidesEncapsulated in Liposomes Comprised of DMTAP:PC:CHOL.

Stability experiments: SQ-20B tumor cells were incubated with a mixtureof radiolabeled-(³²P-γATP) and 10 μM of unlabeled antisense rafoligonucleotide (ATG-AS (SEQ ID NO:1)) either in the liposomeencapsulated form (LE-ATG-AS (SEQ ID NO:1)) (FIG. 19, Lane 1) or freeform (ATG-AS (SEQ ID NO:1)) (FIG. 19, lane 2). The treatment lasted for4 hours at 37° C. in 1% serum containing medium. Immediately followingincubation, cells were washed with phosphate buffered saline (PBS),detached by trypsinization, and collected by centrifugation. The cellpellet was washed twice with PBS, and cells were then lysed in 10 mMTris-HCl, 200 mM NaCl, 1% SDS, 200 μg/ml proteinase K, pH 7.4 for 2hours at 37° C. Oligos were extracted with phenol:chloroform, andaqueous fraction was collected. The samples were normalized for equalradioactivity, and analyzed hy denaturing gel electrophoresis (15%polyacrylamide/7M urea), followed by autoradiography. Data shown in FIG.19 indicate intact ATG-AS (SEQ ID NO:1) oligonucleotide in cells treatedwith LE-ATG-AS (SEQ ID NO:1) (Lane 1), and degraded form of this oligoin cells treated with ATG-AS (SEQ ID NO:1) (Lane 2). Radiolabeledcontrol ATG-AS (SEQ ID NO:1) standard is shown in Lane 3. These datasuggest that encapsulation in the DMTAP:PC:CHOL liposome formulationinhibits degradation of oligos.

Stability experiments: SQ-20B tumor cells were incubated with a mixtureof radiolabeled (³²P-γATP) and 10 μM of unlabeled antisense rafoligonucleotide (ATG-AS (SEQ ID NO:1)) in the liposome encapsulated form(LE-ATG-AS (SEQ ID NO:1)). The treatment lasted for 4 hr at 37° C. in 1%serum containing medium. Following incubation, cells were washed withphosphate buffered saline (PBS), and incubation continued for anadditional 1 hour in 20% serum containing medium. Cells were detached bytrypsinization, and collected by centrifugation. The cell pellet waswashed twice with PBS, and cells were then lysed in 10 mM Tris-HCl, 200mM NaCl, 1% SDS, 15 200 μg/ml proteinase K, pH 7.4 for 2 hours at 37° C.Oligos were extracted with phenol:chloroform, and aqueous fraction wascollected. The samples were normalized for equal radioactivity, andanalyzed by denaturing gel electrophoresis (15% polyacrylamide/7M urea),followed by autoradiography. Data shown in FIG. 20 indicate intactATG-AS (SEQ ID NO:1) oligonucleotide in cells treated with LE-ATG-AS(SEQ ID NO:1) (Lane 1). Radiolabeled control ATG-AS (SEQ ID NO:1)standard is shown in Lane 2. These data further suggest thatoligonucleotide encapsulation in the DMTAP:PC:CHOL liposome formulationprotects oligos and enhances intracellular availability of intactoligos.

In Vivo Results

In Vivo Data

Safety studies of liposomes comprised of DMTAP:PC:CHOL in CD2F1 mice: Todetermine the safety of cationic liposomes comprised of DMTAP:PC:CHOL,these liposome were injected intravenously into male CD2F1 mice (n=5,total lipids equivalent to 5 mg/kg, 15 mg/kg, 25 mg/kg, and 25 mg/kgoligo con), on day 0, 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, and 16. Two ofthe five animals were sacrificed on day 18 for pathology, and theremainder of the animals were sacrificed on day [−] 31. Group bodyweights were monitored throughout the study. As shown in FIG. 21, allanimals survived the treatment and showed weight gain prior to thescheduled termination of this study.

Safety of antisense raf oligodeoxyribonucleotides encapsulated inliposomes comprised of DMTAP:PC:CHOL: To determine the safety ofcationic liposomes comprised of DMTAP:PC:CHOL and encapsulatingantisense raf oligos (LE-ATG-AS (SEQ ID NO:1)), the liposomes containingantisense raf oligos were injected intravenously into male CD2F1 mice(n=5, 5 mg/kg, 15 mg/kg, 25 mg/kg, and 35 mg/kg oligo con), on day 0, 1,3, 4, 6, 7, 9, 10, 12, 13, 15, and 16. Two of the five animals weresacrificed on day 18 for pathological examination, and the remainder ofthe animals was sacrificed on day 31. Group body weights were monitoredthroughout the study. As shown in FIG. 22, all animals survived thetreatment and showed weight gain. These data indicate that LE-ATG-AS(SEQ ID NO:1) composition comprised of DMTAP, PC, CHOL and havingencapsulated therein an antisense raf oligodeoxyribonucleotide is safe.

EXAMPLE 4

Use of raf Antisense Vigo and Chemotherapy in Human Prostate CancerModel

This example relates to the antitumor efficacy of Leraf Aon (SEQ IDNO:1) in combination with cisplatin in athymic nu/nu mice bearing humanprostate cancer (PC-3)-xenografts. Male athymic mice were inoculatedsubcutaneously (s.c) with 5×10⁶ PC-3 cells in 0.2 ml phosphate bufferedsaline/animal. Tumor growth was monitored twice a week until the tumorvolumes were 60 mm³ to 100 mm³′ Animals were randomized into fivetreatment groups indicated above (n=8). Mice were treated with indicateddoses intravenously via the tail vein, and the tumor sizes weremonitored twice a week. Values shown are mean±s.d As shown in FIG. 23,the oligo/cationic liposomal formulation substantially potentated theefficacy of cisplatin as shown by reduced tumor volume relative to thecontrol.

EXAMPLE 5

Use of raf Antisense Oligo with Antichemotherapeutic in Some HumanProstate Cancer Animal Model

The antitumor efficacy of LEraf AON (SEQ ID NO:1) in combination withepirubicin was evaluated in athymic nu/nu mice bearing human prostatecancer (PC-3) xenografts. Male athymic mice were inoculated s.c. with5×10⁶ PC-3 cells in 0.2 ml phosphate buffered saline/animal. Tumorgrowth was monitored twice a week until the tumor volumes were 60 mm³ to100 mm³. Animals were randomized into five treatment groups indicatedabove (n=6). Mice were treated with indicated doses intravenously viathe tail vein, and the tumor sizes were monitored twice a week. Valuesshown are mean±s.d. As shown in FIG. 24, the oligo/cationic system againpotentated efficacy of chemotherapy, particularly epirubicinchemotherapy.

EXAMPLE 6

Use of raf Antisense Oligo with Chemotherapeutic in Human ProstateCancer Animal Model

The antitumor efficacy of LEraf Aon (SEQ ID NO:1) in combination withmitoxantrone was studied in athymic nu/nu mice bearing human prostatecancer (PC-3) xenografts. Male athymic mice were inoculated s.c. with5×10⁶PC-3 cells in 0.2 ml phosphate buffered saline/animal. Tumor growthwas monitored twice a week until the tumor volumes were 60 mm³ to 100mm³. Animals were randomized into five treatment groups indicated above(n=6). Mice were treated with indicated doses intravenously via the tailvein, and the tumor sizes were monitored twice a week. Values shown aremean±s.d. As shown in FIG. 25, the efficacy of chemotherapeutic in thisinstance mitoxantrone again was potentated by the oligo/cationicformulation.

EXAMPLE 7

The antitumor efficacy of a combination of liposome-entrapped rafantisense oligodeoxyribonucleotide (LErafAON) (SEQ ID NO:1) and Gemzar(NDC 0002-7501-01, Gemcitabine HCl, Eli Lilly and Company, Indianapolis,Ind.) was evaluated in athymic mice bearing human pancreatic carcinomaxenografts (Colo357). Athymic mice were inoculated with approximately1.0×10⁶ Colo357 human pancreatic cancer cells. Tumor volumes wereallowed to reach 50 to 100 mm³ in size. Tumor bearing mice were randomlygrouped into five treatment categories (n=5). LErafAON (SEQ ID NO:1)formulation was prepared as described before (Gokhale et al, GeneTherapy 4, 1289–1299, 1997; and Gokhale et al., manuscript inpreparation). Anticancer drug Gemzar (200 mg vials) was purchased fromoncology pharmacy at Georgetown University Hospital and reconstituted innormal saline (12.5 mg/ml). LErafAON (SEQ ID NO:1) (25.0 mg/kg) orGemzar (75.0 mg/kg) was injected intravenously on indicated days. Tumorvolumes were measured twice a week, and tumor sizes (% initial) invarious treatment groups were plotted. As shown in this figure, asignificant tumor growth arrest was noted in the combination treatmentgroup (LErafAON (SEQ ID NO:1) plus Gemzar) as compared to single agenttreatment groups (LErafAON (SEQ ID NO:1) and Gemzar) or control groups(N.S. normal saline, BL, Blank liposomes). Values shown are mean±s.d.The results again show that the cationic/oligo system potentated theanti-tumor efficacy of the chemotherapeutic.

EXAMPLE 8

Use of Antisense raf Oligo and Chemotherapy in Human Pancreatic CancerAnimal Model

The antitumor efficacy of a combination of liposome-entrapped rafantisense oligodeoxyribonucleotide (LErafAON) (SEQ ID NO:1) and Gemzar(NDC 0002-7501-01, Gemcitabine HCl, Eli Lilly and company, Indianapolis,Ind.) was evaluated in athymic mice bearing human pancreatic carcinomaxenografts (Aspc-1). Athymic mice were inoculated with approximately2.5×10⁶ Aspc-1 human pancreatic cancer cells. Tumor volumes were allowedto reach 50 to 100 mm³ in size. Tumor bearing mice were randomly groupedinto five treatment categories (n=5). LErafAON (SEQ ID NO:1) formulationwas prepared as described before (Gokhale et al., Gene Therapy 4,1289–1299, 1997; and Gokhale et al., manuscript in preparation).Anticancer drug Gemzar (200 mg vials) was purchased from oncologypharmacy at Georgetown University Hospital and reconstituted in normalsaline (12.5 mg/ml). LErafAON (SEQ ID NO:1) (25.0 mg/kg) or Gemzar(100.0 mg/kg) was injected intravenously on indicated days. Tumorvolumes were measured twice a week, and tumor sizes (% initial) invarious treatment groups were plotted. As shown in this figure, asignificant tumor growth arrest was noted in the combination treatmentgroup (LErafAON (SEQ ID NO:1) plus Gemzar) as compared to single agenttreatment groups (LErafAON (SEQ ID NO:1) and Gemzar) or control groups(N.S. normal saline, BL, Blank liposomes). Values shown are mean±s.d. Asshown in FIG. 26B, the antitumor activity of the administeredchemotherapeutics (gemzar and gemcitabine HC) was again enhanced as aresult of the oligo/cationic liposomal treatment.

CONCLUSION

The results of examples 4–8 clearly establish that the effects ofchemotherapy, particularly treatment of tumors resistant to chemotherapycan be potentated by the administration of an oligo that targets a geneexpressed by the tumor, e.g., an oncogene such as raf contained in acationic liposomal formulation according to the invention. As thisefficacy has been shown for a variety of different chemotherapeuticsalone or in combination, as well as for different cancers, it isreasonable to expect that this enhancement will be observed in a varietyof different cancers, particularly those resistant to chemotherapy orradiotherapy and with a variety of different chernotherapeutics alone orin combination.

1. A method of chemosensitizing tumor tissue in vivo comprisingadministration of a chemotherapeutic agent and a composition comprisingcationic liposomes, which consist of a cationic lipid,phosphatidylcholine and cholesterol, and having encapsulated therein anoligonucleotide, wherein said oligonucleotide comprises the sequence5′-GTGCTCCATTGATGC-3′ (SEQ ID NO:1) and wherein the chemotherapeuticagent is selected from the group consisting of mitoxantrone, cisplatin,epirubicin, and GEMZAR.
 2. The method of claim 1, wherein theoligonucleotide comprises up to 40 nucleotides and is phosphorothioatedat only the 5′ and 3′ terminal nucleotides.
 3. The method of claim 1,wherein the oligonucleotide comprises up 40 nucleotides and all of itsbases are phosphorothioated.
 4. The method of claim 1, wherein theoligonucleotide comprises up to 40 nucleotides, wherein theoligonucleotide is a chimeric oligonucletide.
 5. The method of claim 1,wherein the oligonucleotide is administered intravenously.
 6. The methodof claim 1, wherein the oligonucleotide is administered directly to thetarget tissue.
 7. The method of claim 1, wherein the oligonucleotide isadministered into the arterial supply to the target tissue.
 8. Themethod of claim 1, wherein said oligonucleotide is an antisense DNA. 9.The method of claim 1, wherein the tumor tissue is caused by cancer. 10.The method of claim 1, wherein the oligonucleotide is administeredbefore or after the chemotherapeutic agent.
 11. The method of claim 1,wherein the oligonucleotide is administered before or after acombination of radiation and the chemotherapeutic agent.
 12. The methodof claim 9, wherein the cancer is selected from the group consisting ofleukemia, lymphoma, myeloma, carcinoma and sarcoma.